Kaposi's sarcoma-associated herpesvirus upregulates angiogenin during infection of human dermal microvascular endothelial cells, which induces 45S rRNA synthesis, antiapoptosis, cell proliferation, migration, and angiogenesis.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is associated with the angioproliferative KS lesions characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth factors, and angiogenic factors. De novo KSHV infection of human microvascular dermal endothelial cells results in increased secretion of several growth factors, cytokines, chemokines, and angiogenic factors, and the multifunctional angiogenic protein angiogenin is one of them. KS tissue sections were positive for angiogenin, highlighting the importance of angiogenin in KS pathogenesis. Examination of KSHV-mediated angiogenin upregulation and secretion and potential outcomes revealed that during infection of primary endothelial cells, KSHV induced a time- and dose-dependent increase in angiogenin gene expression and protein secretion beginning as early as 8 h postinfection and lasting until the fifth day of our observation period. TIVE latently transformed cells (TIVE-LTC) latently infected with KSHV secreted high levels of angiogenin. Angiogenin was also detected in BCBL-1 cells (human B cells) carrying KSHV in a latent state. Significant induction of angiogenin was observed in cells expressing KSHV ORF73 (LANA-1; latent) and ORF74 (lytic) genes alone, and moderate induction was seen with the lytic KSHV ORF50 gene. Angiogenin bound to surface actin, internalized in a microtubule-independent manner, and translocated into the nucleus and nucleolus of infected cells. In addition, it increased 45S rRNA gene transcription, antiapoptosis, and proliferation of infected cells, thus demonstrating the multifunctional nature of KSHV-induced angiogenin. These activities were dependent on angiogenin nuclear translocation, which was inhibited by neomycin. Upregulation of angiogenin led to increased activation of urokinase plasminogen activator and generation of active plasmin, which facilitated the migration of endothelial cells toward chemoattractants, including angiogenin, and chemotaxis was prevented by the inhibition of angiogenin nuclear translocation. Treatment of KSHV-infected cell supernatants with antiangiogenin antibodies significantly inhibited endothelial tube formation, and inhibition of nuclear translocation of angiogenin also blocked the expression of KSHV-induced vascular endothelial growth factor C. Collectively, these results strongly suggest that by increasing infected endothelial cell 45S rRNA synthesis, proliferation, migration, and angiogenesis, KSHV-induced angiogenin could be playing a pivotal role in the pathogenesis of KSHV infection, including a contribution to the angioproliferative nature of KS lesions. Our studies suggested that LANA-1 and vGPCR play roles in KSHV-induced angiogenesis and that the angiogenic potential of vGPCR might also be due to its ability to induce angiogenin.

Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6 and G-protein-coupled receptor regulate angiopoietin-2 expression in lymphatic endothelial cells.

Kaposi's sarcoma (KS) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein-coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm.

The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs). We used these to transduce monocyte-derived dendritic cells (moDCs) isolated from 14 KSHV-seropositive (12 HIV-positive) and 7 KSHV-seronegative (4 HIV-positive) individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle). Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group) was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1]) were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37]) was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1]) was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

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.