Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) Identifies Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Targets in Endothelial Cells

ABSTRACTKaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3′ untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis.IMPORTANCEKS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will help us understand their role in viral oncogenesis. The cross-linking and sequencing of hybrids (CLASH) protocol is a method for unambiguously identifying miRNA targetomes. We developed a streamlined version of CLASH, called quick CLASH (qCLASH). qCLASH requires a lower initial input of cells than for its parent protocol. Additionally, a new fast-growing KSHV-negative endothelial cell line, named TIVE-EX-LTC cells, was established. qCLASH was performed on TIVE-EX-LTC cells latently infected with wild-type (WT) KSHV or a mutant virus lacking miR-K12-11/11*. A number of novel targets of KSHV miRNAs were identified, including targets of miR-K12-11, the ortholog of the cellular oncogenic miRNA (oncomiR) miR-155. Many of the miRNA targets were involved in processes related to oncogenesis, such as glycolysis, apoptosis, and cell cycle control.

Download Full-text

HIV-1 Tat Regulates Endothelial Cell Cycle Progression via Activation of the Ras/ERK MAPK Signaling Pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0717 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1985-1994 ◽

Cited By ~ 47

Author(s):

Elena Toschi ◽

Ilaria Bacigalupo ◽

Raffaele Strippoli ◽

Chiara Chiozzini ◽

Anna Cereseto ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Kaposi's Sarcoma ◽

Cell Cycle Progression ◽

Kaposi’S Sarcoma ◽

Mapk Signaling ◽

Small Gtpases ◽

Mitogen Activated Protein Kinase ◽

Endothelial Cell Proliferation ◽

Hiv 1

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the α5β1 and αvβ3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.

Download Full-text

Activation of NF-κB by the Human Herpesvirus 8 Chemokine Receptor ORF74: Evidence for a Paracrine Model of Kaposi's Sarcoma Pathogenesis

Journal of Virology ◽

10.1128/jvi.75.18.8660-8673.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8660-8673 ◽

Cited By ~ 126

Author(s):

Shibani Pati ◽

Marielle Cavrois ◽

Hong-Guang Guo ◽

James S. Foulke ◽

Jynho Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Kaposi's Sarcoma ◽

Vascular Endothelial Cells ◽

Inositol Phosphate ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lymphoid Cells ◽

Human Herpesvirus 8 ◽

Herpesvirus 8 ◽

Latently Infected

ABSTRACT Infection with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is necessary for the development of KS. The HHV-8 lytic-phase gene ORF74 is related to G protein-coupled receptors, particularly interleukin-8 (IL-8) receptors. ORF74 activates the inositol phosphate/phospholipase C pathway and the downstream mitogen-activated protein kinases, JNK/SAPK and p38. We show here that ORF74 also activates NF-κB independent of ligand when expressed in KS-derived HHV-8-negative endothelial cells or primary vascular endothelial cells. NF-κB activation was enhanced by the chemokine GROα, but not by IL-8. Mutation of Val to Asp in the ORF74 second cytoplasmic loop did not affect ligand-independent signaling activity, but it greatly increased the response to GROα. ORF74 upregulated the expression of NF-κB-dependent inflammatory cytokines (RANTES, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Supernatants from transfected KS cells activated NF-κB signaling in untransfected cells and elicited the chemotaxis of monocytoid and T-lymphoid cells. Expression of ORF74 conferred on primary endothelial cells a morphology that was strikingly similar to that of spindle cells present in KS lesions. Taken together, these data, demonstrating that ORF74 activates NF-κB and induces the expression of proangiogenic and proinflammatory factors, suggest that expression of ORF74 in a minority of cells in KS lesions could influence uninfected cells or latently infected cells via autocrine and paracrine mechanisms, thereby contributing to KS pathogenesis.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces Expression of the Helix-Loop-Helix Protein Id-1 in Human Endothelial Cells

Journal of Virology ◽

10.1128/jvi.77.10.5975-5984.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5975-5984 ◽

Cited By ~ 37

Author(s):

Jun Tang ◽

Gabriel M. Gordon ◽

Maike G. Müller ◽

Madhu Dahiya ◽

Kimberly E. Foreman

Keyword(s):

Cell Cycle ◽

Endothelial Cells ◽

Tumor Cells ◽

Cell Cycle Regulation ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Helix Loop Helix ◽

Cycle Regulation

ABSTRACT Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) is a gamma-2 herpesvirus believed to be the etiologic agent responsible for KS. The pathogenesis of this potentially life-threatening neoplasm is complex and unclear, and it is currently unknown how KSHV causes KS. Id (named for inhibitor of DNA binding or inhibitor of differentiation) proteins were identified in 1990 and found to be naturally occurring dominant-negative inhibitors of basic helix-loop-helix transcription factors. Id-1, the most well-studied member of this family, has since been shown to play a key role in several biological systems including cellular differentiation, cell cycle regulation, and tumorigenesis. In this report, we demonstrate that Id-1 is expressed at high levels in KS tumor cells both in vitro and in vivo but is expressed at relatively modest levels in endothelial cells (ECs), the likely precursor of the KS tumor cell. Infection of precursor cells with KSHV may be responsible for this enhanced expression, as KSHV infection induced Id-1 27-fold in ECs under our experimental conditions. Furthermore, we demonstrate that the KSHV-encoded latency-associated nuclear antigen (LANA) protein appears to be involved. Expression of LANA in ECs resulted in Id-1 induction that was almost identical to the induction seen with KSHV-infected ECs. These results demonstrate the expression of Id-1 in KS tumor cells and indicate the KSHV LANA protein may be, at least in part, responsible. This may be an important mechanism by which KSHV allows KS tumor cells to escape normal cell cycle regulation and enhances their proliferation.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Mutants Modulate Cancer Hallmark Phenotypic Differences in Human Endothelial Cells

Journal of Virology ◽

10.1128/jvi.02022-20 ◽

2021 ◽

Vol 95 (7) ◽

Author(s):

Lauren A. Gay ◽

Daniel Stribling ◽

Peter C. Turner ◽

Rolf Renne

Keyword(s):

Endothelial Cells ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Wild Type ◽

Human Endothelial Cells ◽

Tubule Formation ◽

Phenotypic Changes ◽

Individual Mirnas ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi’s sarcoma (KS) results from the transformation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected endothelial cells. The contribution of the KSHV microRNAs (miRNAs) to the process of oncogenesis in endothelial cells has not been fully elucidated. To better understand the contributions of individual miRNAs to oncogenesis-related cellular phenotypes, we used KSHV miRNA knockout mutants, each lacking one of the 12 miRNA genes. An additional mutant lacked all miRNAs. Since KSHV infection causes a variety of phenotypic changes in endothelial cells, we tested the mutants for their ability to effect such changes in telomerase-immortalized vein endothelial (TIVE) cells infected with each of the mutant viruses. Wild type- and mutant-infected as well as uninfected cells were evaluated for perturbations to proliferation, migration, tubule formation, and glycolysis. We found broad variation between the different viruses in these aspects. With respect to proliferation rate, ΔmiR-K12-3, ΔmiR-K12-8, and ΔmiR-K12-11 showed significant impairment. Cells infected with ΔmiR-K12-11 had reduced migration. In tubule formation, the ΔmiR-K12-5, -6, and -7 viruses were deficient. At the same time, cells infected with the ΔmiR-K12-10 virus showed dysregulated glycolysis. By combining these observations with previously published KSHV miRNA targetome lists from ribonomics data, we were able to functionally validate a number of new miRNA targets in specific pathways. As proof of concept, miR-K12-3 was shown to target cathepsin D, a strong promoter of apoptosis. Taken together, the results demonstrate that KSHV miRNAs play different roles in inducing the phenotypic changes that are characteristic of transformed cells. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma (KS). The contribution of KSHV microRNAs (miRNAs) to oncogenesis is not fully understood. This is particularly true for human endothelial cells, the cell type from which KS tumors are derived. Here, we used a panel of KSHV miRNA knockout viruses to shed light on the roles of individual miRNAs in the process of transformation. Latently infected endothelial cells were studied for phenotypic changes related to cancer, including proliferation, migration, angiogenesis, glycolysis, and apoptosis. The mutant-infected cell lines displayed a wide range of phenotypes in these selected measures of oncogenesis, which differed from those of wild-type-infected cells and from each other. These results indicate that KSHV miRNAs contribute to different aspects of oncogenesis and that each one has a unique role to play.

Download Full-text

De Novo Infection and Serial Transmission of Kaposi's Sarcoma-Associated Herpesvirus in Cultured Endothelial Cells

Journal of Virology ◽

10.1128/jvi.76.5.2440-2448.2002 ◽

2002 ◽

Vol 76 (5) ◽

pp. 2440-2448 ◽

Cited By ~ 147

Author(s):

Michael Lagunoff ◽

Jill Bechtel ◽

Eleni Venetsanakos ◽

Anne-Marie Roy ◽

Nancy Abbey ◽

...

Keyword(s):

Endothelial Cells ◽

Kaposi's Sarcoma ◽

Cytopathic Effect ◽

De Novo ◽

Kaposi’S Sarcoma ◽

Serial Passage ◽

Lytic Cycle ◽

Microvascular Endothelial Cells ◽

Latently Infected ◽

Microvascular Endothelial

ABSTRACT Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is central to the pathogenesis of the endothelial neoplasm Kaposi's sarcoma (KS) and is also linked to the rare B-cell tumor known as primary effusion lymphoma (PEL). Latently infected PEL cell lines can be induced to enter the lytic cycle and produce KSHV virions. However, such cells do not support de novo infection or serial propagation of KSHV. These limitations have prevented the development of systems for the genetic analysis of KSHV and have impeded a deeper understanding of KS pathogenesis. Here we show that human dermal microvascular endothelial cells immortalized by expression of telomerase can be readily infected by KSHV virions produced by PEL cells. Infection is predominantly latent, but a small subpopulation enters the lytic cycle spontaneously. Phorbol ester (tetradecanoyl phorbol acetate [TPA]) treatment of latently infected cells leads to enhanced induction of lytic KSHV replication, resulting in foci of cytopathic effect. There is no cytopathic effect or viral DNA expansion when infected TIME cells (telomerase-immortalized microvascular endothelial cells) are TPA induced in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus replication. Supernatants from phorbol-induced cultures transfer latent KSHV infection to uninfected cells, which can likewise be induced to undergo lytic replication by TPA treatment, and the virus can be further serially transmitted. Serial passage of the virus in TIME cells is completely inhibited when TPA treatment is done in the presence of PAA. Latently infected endothelial cells do not undergo major morphological changes or growth transformation, and infection is lost from the culture upon serial passage. This behavior faithfully recapitulates the behavior of spindle cells explanted from primary KS biopsies, strongly supporting the biological relevance of this culture system. These findings suggest that either the stability or the growth-deregulatory potential of the KSHV latency program in endothelial cells is more limited than might be predicted by analogy with other oncogenic viruses.

Download Full-text

A CRISPR-Cas9 screen identifies mitochondrial translation as an essential process in latent KSHV infection of human endothelial cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2011645117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28384-28392

Author(s):

Daniel L. Holmes ◽

Daniel T. Vogt ◽

Michael Lagunoff

Keyword(s):

Endothelial Cells ◽

Mitochondrial Respiration ◽

Kaposi's Sarcoma ◽

Latent Infection ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Etiologic Agent ◽

Mitochondrial Translation ◽

Enhanced Sensitivity ◽

Latently Infected

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus Glycoproteins B and K8.1 Regulate Virion Egress and Synthesis of Vascular Endothelial Growth Factor and Viral Interleukin-6 in BCBL-1 Cells

Journal of Virology ◽

10.1128/jvi.01889-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1704-1714 ◽

Cited By ~ 16

Author(s):

R. Subramanian ◽

I. Sehgal ◽

O. D'Auvergne ◽

K. G. Kousoulas

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cell ◽

Growth Factor ◽

Interleukin 6 ◽

Kaposi's Sarcoma ◽

Substantial Reduction ◽

Kaposi’S Sarcoma ◽

Vascular Endothelial ◽

Wild Type ◽

Endothelial Growth Factor

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) viral glycoproteins play important roles in the infectious life cycle and have been implicated in KSHV-associated endothelial cell transformation, angiogenesis, and KS-induced malignancies. KSHV-associated primary effusion lymphomas (PELs) secrete high levels of vascular endothelial growth factor (VEGF) and viral interleukin-6 (vIL-6) in vitro and VEGF, vIL-6, and basic-fibroblast growth factor (b-FGF) in mouse xenografts. KSHV-encoded glycoproteins B (gB) and K8.1 stimulate VEGF secretion, most likely mediated by direct or indirect binding to cell surface receptors, including the gB-specific αVβ3 and α3β1 integrins. In this study, the short interfering RNA (siRNA)-mediated inhibition of either gB or K8.1 transcription by anti-gB or -K8.1 siRNAs caused a substantial reduction in virion egress and a decrease in both vIL-6 and VEGF production. Similarly, the treatment of BCBL-1 cells with anti-gB or anti-K8.1 antibodies caused a substantial reduction in vIL-6 and VEGF production. Codon-optimized versions of either wild-type gB, mutant gB having the RGD amino acid motif changed to RAA, or K8.1 efficiently rescued virion egress and VEGF and vIL-6 production. These results suggest that the binding of gB via its RGD motif to integrin receptors was not responsible for the observed gB-associated regulation of VEGF and vIL-6 transcription. Conditioned medium collected from BCBL-1 cells transfected with anti-gB and anti-K8.1 siRNAs or treated with anti-gB and anti-K8.1 antibodies exhibited a significantly reduced ability to induce the formation of the capillary network of endothelial cells compared to the ability of medium from mock-infected BCBl-1 cells. Furthermore, medium obtained from BCBL-1 cells expressing smaller amounts of gB and K8.1 produced a substantial reduction in endothelial cell migration in a vertical migration assay compared to that of control medium containing wild-type levels of gB and K8.1. These results suggest a functional linkage between gB/K8.1 synthesis and VEGF/vIL-6 transcriptional regulation via paracrine and/or autocrine signaling pathways.

Download Full-text

3D culture conditions support Kaposi’s sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells

Journal of Molecular Medicine ◽

10.1007/s00109-020-02020-8 ◽

2021 ◽

Vol 99 (3) ◽

pp. 425-438

Author(s):

Tatyana Dubich ◽

Anne Dittrich ◽

Kristine Bousset ◽

Robert Geffers ◽

Guntram Büsche ◽

...

Keyword(s):

Cell Culture ◽

Endothelial Cells ◽

Endothelial Cell ◽

Kaposi's Sarcoma ◽

3D Cell Culture ◽

Kaposi’S Sarcoma ◽

Culture Conditions ◽

List Type ◽

Infection Frequency

Abstract Kaposi’s sarcoma–associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi’s sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman’s disease). While in patients with late stage of Kaposi’s sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. Key messages In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance

Download Full-text

Induction of the Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1004882107 ◽

2010 ◽

Vol 107 (23) ◽

pp. 10696-10701 ◽

Cited By ~ 123

Author(s):

T. Delgado ◽

P. A. Carroll ◽

A. S. Punjabi ◽

D. Margineantu ◽

D. M. Hockenbery ◽

...

Keyword(s):

Endothelial Cells ◽

Warburg Effect ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Latently Infected ◽

The Warburg Effect

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications

Journal of Virology ◽

10.1128/jvi.00873-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1759-1776 ◽

Cited By ~ 47

Author(s):

Ramu Sivakumar ◽

Neelam Sharma-Walia ◽

Hari Raghu ◽

Mohanan Valiya Veettil ◽

Sathish Sadagopan ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Endothelial Cell ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Angiogenic Factors ◽

Vascular Endothelial ◽

Kshv Infection ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma (KS), a vascular tumor associated with human immunodeficiency virus type 1 infection, is characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth and angiogenic factors, and angiogenesis. KS spindle cells are believed to be of the lymphatic endothelial cell (LEC) type. Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) is etiologically linked to KS, and in vitro KSHV infection of primary human dermal microvascular endothelial cells (HMVEC-d) is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, sustained induction of NF-κB and several cytokines, and growth and angiogenic factors. KSHV induced robust vascular endothelial growth factor A (VEGF-A) and VEGF-C gene expression as early as 30 min postinfection (p.i.) in serum-starved HMVEC-d, which was sustained throughout the observation period of 72 h p.i. Significant amounts of VEGF-A and -C were also detected in the culture supernatant of infected cells. VEGF-A and -C were also induced by UV-inactivated KSHV and envelope glycoprotein gpK8.1A, thus suggesting a role for virus entry stages in the early induction of VEGF and requirement of KSHV viral gene expression for sustained induction. Exogenous addition of VEGF-A and -C increased KSHV DNA entry into target cells and moderately increased latent ORF73 and lytic ORF50 promoter activation and gene expression. KSHV infection also induced the expression of lymphatic markers Prox-1 and podoplanin as early as 8 h p.i., and a paracrine effect was seen in the neighboring uninfected cells. Similar observations were also made in the pure blood endothelial cell (BEC)-TIME cells, thus suggesting that commitment to the LEC phenotype is induced early during KSHV infection of blood endothelial cells. Treatment with VEGF-C alone also induced Prox-1 expression in the BEC-TIME cells. Collectively, these studies show that the in vitro microenvironments of KSHV-infected endothelial cells are enriched, with VEGF-A and -C molecules playing key roles in KSHV biology, such as increased infection and gene expression, as well as in angiogenesis and lymphangiogenesis, thus recapitulating the microenvironment of early KS lesions.

Download Full-text