Glycyrrhizic acid alters Kaposi sarcoma-associated herpesvirus latency, triggering p53-mediated apoptosis in transformed B lymphocytes.

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with all clinical forms of Kaposi sarcoma and several lymphoproliferative disorders. Like other herpesviruses, KSHV becomes latent in the infected cells, expressing only a few genes that are essential for the establishment and maintenance of its latency and for the survival of the infected cells. Inhibiting the expression of these latent genes should lead to eradication of herpesvirus infection. All currently available drugs are ineffective against latent infection. Here we show, for the first time to our knowledge, that latent infection with KSHV in B lymphocytes can be terminated by glycyrrhizic acid (GA), a triterpenoid compound earlier shown to inhibit the lytic replication of other herpesviruses. We demonstrate that GA disrupts latent KSHV infection by downregulating the expression of latency-associated nuclear antigen (LANA) and upregulating the expression of viral cyclin and selectively induces cell death of KSHV-infected cells. We show that reduced levels of LANA lead to p53 reactivation, an increase in ROS, and mitochondrial dysfunction, which result in G1 cell cycle arrest, DNA fragmentation, and oxidative stress-mediated apoptosis. Latent genes are involved in KSHV-induced oncogenesis, and strategies to interfere with their expression might prove useful for eradicating latent KSHV infection and have future therapeutic implications.

Natural killer cells are not infected by Kaposi's sarcoma-associated herpesvirus in vivo, and natural killer cell counts do not correlate with the risk of developing Kaposi's sarcoma.

Although the innate immune system is implicated in the control of Kaposi's sarcoma (KS), the risk of developing KS is not associated with the nadir natural killer (NK) cell count, and NK cell counts do not significantly increase or decrease during KS resolution. KS-associated herpesvirus replication was not demonstrated in vivo or in vitro within NK cells, suggesting that NK cells do not contribute to the resolution of KS. Their role appears limited to events occurring during early infection.

Detection and quantitation of Kaposi's sarcoma-associated herpesvirus (KSHV) by a single competitive-quantitative polymerase chain reaction.

Kaposi's sarcoma-associated herpesvirus is a novel herpesvirus linked to AIDS-related neoplasms. Currently it is difficult to evaluate the number of virions in viral preparation or in samples obtained from patients with Kaposi's sarcoma (KS), since no protocol for determining the plaque forming units of KSHV exists. We constructed a fragment of a different size than the target viral DNA to carry out a competitive-quantitative PCR. Both fragment and viral DNA were added to a single PCR reaction to compete for the same set of primers. By knowing the amount of the competitor added to the reaction, we could determine the number of viral DNA molecules. We used this assay successfully to detect and quantify KSHV genomes from KS skin biopsies and pleural effusion lymphoma, and from different viral preparations. To date, this is the most convenient and economic method that allows an accurate and fast viral detection/quantitation with a single PCR.

Interaction of Adenovirus E1A with the HHV8 Promoter of Latent Genes: E1A Proteins are Able to Activate the HHV-8 LANAp in MV3 Reporter Cells.

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, body cavity-based lymphoma, and Castleman's disease. Adenoviral (Ad) E1A proteins regulate the activity of cellular and viral promoters/enhancers and transcription factors and can suppress tumorigenicity of human cancers. As (i) HHV-8 and Ad may co-exist in immunocompromised patients and (ii) E1A might be considered as therapeutic transgene for HHV-8-associated neoplasms we investigated whether the promoter of the latency-associated nuclear antigen (LANAp) controlling expression of vCyclin, vFLIP, and LANA proteins required for latent type infection is regulated by E1A. Transfection experiments in MV3 melanoma cells revealed activation of the LANAp by Ad5 E1A constructs containing an intact N terminus (aa 1-119). In particular, an Ad12 E1A mutant, Spm2, lacking six consecutive alanine residues in the "spacer" region activated the HHV-8 promoter about 15-fold compared to vector controls. In summary, we report the activation of the LANAp by E1A as a novel interaction of E1A with a viral promoter. These data may have relevance for the management of viral infections in immunocompromised patients. A role for E1A as a therapeutic in this context remains to be defined.

Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells.

Kaposi sarcoma-associated herpesvirus (KSHV) is etiologically linked to Kaposi sarcoma (KS), a tumor genetically akin to lymphatic endothelial cells (LECs). We obtained the immune transcriptional signature of KS and used KSHV-infected LECs (KLECs) as an in vitro model to determine the effects of KSHV on transcription and expression of genes involved in immunity. The antigen presentation, interferon (IFN) response, and cytokine transcriptomes of KLECs resemble those of KS. Transcription of genes involved in class I presentation is increased in KS and after infection of LECs, but MHC-I and ICAM-1 surface expression are down-regulated in KLECs. Inhibition of IFN induction of MHC-I transcription indicates that KSHV regulates MHC-I transcription. We show that MHC-I transcription is regulated by the KSHV-encoded viral FLICE inhibitory protein (vFLIP) and by viral IFN regulatory factor 1 (vIRF1). vFLIP up-regulates MHC-I and ICAM-1 through activation of NF-kappaB and stimulates T-cell proliferation, revealing a mechanism to prevent uncontrolled viral dissemination. In contrast, vIRF1 inhibits basal and IFN- and vFLIP-induced MHC-I transcription and surface expression through its interaction with the transcriptional coactivator p300, contributing to immune evasion. We propose that regulation of MHC-I by vFLIP and vIRF1 plays a crucial role in the host-pathogen equilibrium.

Human herpesvirus-8-transformed endothelial cells have functionally activated vascular endothelial growth factor/vascular endothelial growth factor receptor.

Kaposi's sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi's sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression.

Transcriptional downregulation of ORF50/Rta by methotrexate inhibits the switch of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 from latency to lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cellular dihydrofolate reductase (DHFR) homologue. Methotrexate (MTX), a potent anti-inflammatory agent, inhibits cellular DHFR activity. We investigated the effect of noncytotoxic doses of MTX on latency and lytic KSHV replication in two KSHV-infected primary effusion lymphoma cell lines (BC-3 and BC-1) and in MTX-resistant BC-3 cells (MTX-R-BC-3 cells). Treatment with MTX completely prevented tetradecanoyl phorbol acetate-induced viral DNA replication and strongly decreased viral lytic transcript levels, even in MTX-resistant cells. However, the same treatment had no effect on transcription of cellular genes and KSHV latent genes. One of the lytic transcripts inhibited by MTX, ORF50/Rta (open reading frame), is an immediate-early gene encoding a replication-transcription activator required for expression of other viral lytic genes. Therefore, transcription of genes downstream of ORF50/Rta was inhibited, including those encoding the viral G-protein-coupled receptor (GPCR), viral interleukin-6, and K12/kaposin, which have been shown to be transforming in vitro and oncogenic in mice. Resistance to MTX has been documented in cultured cells and also in patients treated with this drug. However, MTX showed an inhibitory activity even in MTX-R-BC-3 cells. Two currently available antiherpesvirus drugs, cidofovir and foscarnet, had no effect on the transcription of these viral oncogenes and ORF50/Rta. MTX is the first example of a compound shown to downregulate the expression of ORF50/Rta and therefore prevent viral transforming gene transcription. Given that the expression of these genes may be important for tumor development, MTX could play a role in the future management of KSHV-associated malignancies.