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.     

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.     

Oxidative stress induces reactivation of Kaposi's sarcoma-associated herpesvirus and death of primary effusion lymphoma cells

Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) cells are predominantly infected with latent Kaposi's sarcoma-associated herpesvirus (KSHV), presenting a barrier to the destruction of tumor cells. Latent KSHV can be reactivated to undergo lytic replication. Here we report that in PEL cells, oxidative stress induced by upregulated reactive oxygen species (ROS) can lead to KSHV reactivation or cell death. ROS are upregulated by NF-κB inhibition and are required for subsequent KSHV reactivation. Disruption of the intracellular redox balance through depletion of the antioxidant glutathione or inhibition of the antioxidant enzyme catalase also induces KSHV reactivation, suggesting that hydrogen peroxide induces reactivation. In addition, p38 signaling is required for KSHV reactivation induced by ROS. Furthermore, treatment of PEL cells with a higher concentration of the NF-κB inhibitor than that used for inducing KSHV reactivation further upregulates ROS and induces massive cell death. ROS, but not p38 signaling, are required for PEL cell death induced by NF-κB inhibition as well as by glutathione depletion. Importantly, anticancer drugs, such as cisplatin and arsenic trioxide, also induce KSHV reactivation and PEL cell death in a ROS-dependent manner. Our study thus establishes a critical role for ROS and oxidative stress in the regulation of KSHV reactivation and PEL cell death. Disrupting the cellular redox balance may be a potential strategy for treating KSHV-associated lymphoma.     

A novel role of hydrogen peroxide in Kaposi sarcoma-associated herpesvirus reactivation

Reactivation of Kaposi sarcoma-associated herpesvirus (KSHV) from latency for lytic replication plays a pivotal role in the development of KS tumors. However, the physiological factors of KSHV reactivation in KS patients remain undefined. Two recent studies independently discovered that the reactive oxygen species (ROS) H2O2 induces KSHV reactivation in latently infected cells, which can be inhibited by H2O2-specific antioxidants. H2O2 not only directly induces KSHV reactivation but also is involved in spontaneous lytic replication as well as reactivation stimulated by TPA, hypoxia, and cytokines. Furthermore, in a xenograft-based primary effusion lymphoma (PEL) mouse model, in vivo KSHV reactivation is also H2O2-dependent and can be suppressed by antioxidants. Mechanistically, H2O2 primarily activates the MAPK pathways to induce viral lytic gene expression and replication. This new finding defines a novel role of H2O2 in KS tumorigenesis and highlights great potentials of using antioxidants and anti-inflammatory drugs for the prevention and treatment of KS tumors.     

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.     

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.     

Effect of extremely low frequency electromagnetic fields (ELF-EMF) on Kaposi's sarcoma-associated herpes virus in BCBL-1 cells

Association between extremely low frequency electromagnetic fields (ELF-EMF) and human cancers is controversial, and few studies have been conducted on their influence on oncogenic viruses. We studied the effects of 1 mT, 50 Hz sine waves, applied for 24-72 h, on Kaposi's sarcoma (KS)-associated herpesvirus (KSHV or HHV-8) in BCBL-1, a latently infected primary effusion lymphoma (PEL) cell line. ELF-EMF exposure did not affect the growth and viability of BCBL-1 cells, either stimulated or not with TPA. The total amount of KSHV DNA detected in ELF-EMF exposed cultures not stimulated with TPA did not differ from that of the unexposed controls (P = ns). However, in the presence of TPA stimulation, total KSHV DNA content was found higher in ELF-EMF exposed than in control BCBL-1 cultures (P = .024) at 72 h exposure, but not earlier. Viral DNA increase significantly correlated with increased mean fluorescence intensity/cell for the lytic antigen gp K8.1A/B (P < .01), but not with percentage of gp K8.1A/B-positive cells or of cells containing virions. Viral progeny produced under ELF-EMF exposure consisted mainly of defective viral particles.     

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the cis-acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that cis-acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.     

Functional p53 signaling in Kaposi's sarcoma-associated herpesvirus lymphomas: implications for therapy

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.     

Cdk1 inhibition induces mutually inhibitory apoptosis and reactivation of Kaposi's sarcoma-associated herpesvirus

Primary effusion lymphoma (PEL) cells are predominantly infected by the latent form of Kaposi's sarcoma-associated herpesvirus (KSHV), with virus reactivation occurring in a small percentage of cells. Latency enables KSHV to persist in the host cell and promotes tumorigenesis through viral gene expression, thus presenting a major barrier to the elimination of KSHV and the treatment of PEL. Therefore, it is important to identify cellular genes that are essential for PEL cell survival or the maintenance of KSHV latency. Here we report that cyclin-dependent kinase 1 (Cdk1) inhibition can induce both apoptosis and KSHV reactivation in a population of PEL cells. Caspases, but not p53, are required for PEL cell apoptosis induced by Cdk1 inhibition. p38 kinase is activated by Cdk1 inhibition and mediates KSHV reactivation. Interestingly, upon Cdk1 inhibition, KSHV is reactivated predominantly in the nonapoptotic subpopulation of PEL cells. We provide evidence that this is due to mutual inhibition between apoptosis and KSHV reactivation. In addition, we found that KSHV reactivation activates protein kinase B (AKT/PKB), which promotes cell survival and facilitates KSHV reactivation. Our study thus establishes a key role for Cdk1 in PEL cell survival and the maintenance of KSHV latency and reveals a multifaceted relationship between KSHV reactivation and PEL cell apoptosis.