Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.     

Kaposi's sarcoma-associated herpesvirus-induced upregulation of the c-kit proto-oncogene,as identified by gene expression profiling,is essential for the transformation of endothelial cells

Kaposi's sarcoma (KS), the most frequent malignancy afflicting AIDS patients, is characterized by spindle cell formation and vascularization. Infection with KS-associated herpesvirus (KSHV) is consistently observed in all forms of KS. Spindle cell formation can be replicated in vitro by infection of dermal microvascular endothelial cells (DMVEC) with KSHV. To study the molecular mechanism of this transformation, we compared RNA expression profiles of KSHV-infected and mock-infected DMVEC. Induction of several proto-oncogenes was observed, particularly the receptor tyrosine kinase c-kit. Consistent with increased c-Kit expression, KHSV-infected DMVEC displayed enhanced proliferation in response to the c-Kit ligand, stem cell factor (SCF). Inhibition of c-Kit activity with either a pharmacological inhibitor of c-Kit (STI 571) or a dominant-negative c-Kit protein reversed SCF-dependent proliferation. Importantly, inhibition of c-Kit signal transduction reversed the KSHV-induced morphological transformation of DMVEC. Furthermore, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation. Together, these data demonstrate an essential role for c-Kit in KS tumorigenesis and reveal a target for pharmacological intervention.     

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

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.     

Surface downregulation of major histocompatibility complex class I,PE-CAM,and ICAM-1 following de novo infection of endothelial cells with Kaposi's sarcoma-associated herpesvirus

Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.     

Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi's sarcoma-associated herpesvirus latency in vitro and in vivo

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.     

Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma and several other malignancies. The lack of an efficient infection system has impeded the understanding of KSHV-related pathogenesis. A genetic approach was used to isolate infectious KSHV. Recombinant bacteria artificial chromosome (BAC) KSHV containing hygromycin resistance and green fluorescent protein (GFP) markers was generated by homologous recombination in KSHV-infected BCBL-1 cells. Recombinant KSHV genomes from cell clones that were resistant to hygromycin, expressed GFP, and produced infectious virions after induction with tetradecanoyl phorbol acetate (TPA) were rescued in Escherichia coli and reconstituted in 293 cells. Several 293 cell lines resulting from infection with recombinant virions induced from a full-length recombinant KSHV genome, named BAC36, were obtained. BAC36 virions established stable latent infection in 293 cells, harboring 1 to 2 copies of viral genome per cell and expressing viral latent proteins, with approximately 0.5% of cells undergoing spontaneous lytic replication, which is reminiscent of KSHV infection in Kaposi's sarcoma tumors. TPA treatment induced BAC36-infected 293 cell lines into productive lytic replication, expressing lytic proteins and producing virions that efficiently infected normal 293 cells with a approximately 50% primary infection rate. BAC36 virions were also infectious to HeLa and E6E7-immortalized human endothelial cells. Since BAC36 can be efficiently shuttled between bacteria and mammalian cells, it is useful for KSHV genetic analysis. The feasibility of the system was illustrated through the generation of a KSHV mutant with the vIRF gene deleted. This cellular model is useful for the investigation of KSHV infection and pathogenesis.     

De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells

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.     

Inflammatory cytokines inhibit Kaposi's sarcoma-associated herpesvirus lytic gene transcription in in vitro-infected endothelial cells

The response of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) to inflammatory cytokine treatment of experimentally infected endothelial cells was investigated. The cytokines inhibited spontaneous KSHV lytic gene expression but not the level of infection. The data suggest that if inflammatory cytokines present in KS lesions contribute to KSHV pathogenesis, they do so in part by promoting latent KSHV infection of the endothelial cells.     

Persistent activation of STAT3 by latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and plasmablastic multicentric Castleman's disease. STAT3 has been shown to be important for the maintenance of primary effusion lymphoma cells in culture and is chronically activated in many tumor cell lines. However, little is known about the role of KSHV in the activation of STAT3 or the role of STAT3 in KS tumors. We demonstrate that STAT3 is activated by KSHV infection of endothelial cells, the KS tumor cell type, in a biphasic fashion. Viral binding and entry activate STAT3 in the first 2 h after infection, but this activation dissipates by 4 h postinfection. By 12 h after KSHV infection, concomitant with the expression of latent genes, STAT3 is once again activated, and this activation persists for as long as latent infection is maintained. Activated STAT3 translocates to the nucleus, where it can bind to STAT3-specific DNA elements and can activate STAT3-dependent promoter activity. Conditioned medium from KSHV-infected endothelial cells is able to transiently activate STAT3, indicating the involvement of a secreted factor and that a latency-associated factor in KSHV-infected cells is necessary for sustained activation. KSHV upregulates gp130 receptor expression, and both gp130 and JAK2 are required for the activation of STAT3. However, neither human nor viral interleukin-6 is required for STAT3 activation. Persistent activation of the oncogenic signal transducer, STAT3, by KSHV may play a critical role in the viral pathogenesis of Kaposi's sarcoma, as well as in primary effusion lymphomas.     

Propranolol Decreases Proliferation of Endothelial Cells Transformed by Kaposi's Sarcoma-Associated Herpesvirus and Induces Lytic Viral Gene Expression

Kaposi''s sarcoma (KS) is common in Africa, but economic constraints hinder successful treatment in most patients. Propranolol, a generic β-adrenergic antagonist, decreased proliferation of KS-associated herpesvirus (KSHV)-infected cells. Downregulation of cyclin A2 and cyclin-dependent kinase 1 (CDK1) recapitulated this phenotype. Additionally, propranolol induced lytic gene expression in association with downregulation of CDK6. Thus, propranolol has diverse effects on KSHV-infected cells, and this generic drug has potential as a therapeutic agent for KS.     

Activation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) lytic replication by human cytomegalovirus

The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) and neo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.     

Kaposi's sarcoma-associated herpesvirus disrupts adherens junctions and increases endothelial permeability by inducing degradation of VE-cadherin

Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial cells caused by KS-associated herpesvirus (KSHV) infection. Aberrant vascular permeability is a hallmark of KS manifested as multifocal edematous skin and visceral lesions with dysregulated angiogenesis and vast inflammatory infiltrations. In this study, we showed that KSHV infection increased the permeability of confluent endothelial monolayers to serum albumin, blood-derived cells, KSHV-infected cells, and KSHV virions. KSHV-induced permeability was associated with the disruption of adherens junctions and the degradation of vascular endothelial cadherin (VE-cadherin) protein. Both the inactivation of KSHV virions by UV irradiation and the blockage of de novo protein synthesis with cycloheximide failed to reverse the KSHV-induced disruption of adherens junctions. However, soluble heparin that blocked KSHV entry into cells completely inhibited KSHV-induced permeability. Furthermore, the KSHV-induced degradation of VE-cadherin was dose dependent on the internalized virus particles. Together, these results indicate that KSHV infection induces vascular permeability by inducing VE-cadherin degradation during virus entry into cells. KSHV-induced aberrant vascular permeability could facilitate virus spread, promote inflammation and angiogenesis, and contribute to the pathogenesis of KSHV-induced malignancies.     

Lytic replication-defective Kaposi's sarcoma-associated herpesvirus: potential role in infection and malignant transformation

Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G(0)/G(1) apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.