A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We found that beta-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated beta-catenin accumulation; LANA itself upregulated expression of beta-catenin in transfected cells. LANA stabilizes beta-catenin by binding to the negative regulator GSK-3beta, causing a cell cycle-dependent nuclear accumulation of GSK-3beta. The LANA C terminus contains sequences similar to the GSK-3beta-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3beta or stabilize beta-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3beta can therefore be a source of beta-catenin dysregulation in human cancers.     

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.     

Ets-1-dependent expression of vascular endothelial growth factor receptors is activated by latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus through interaction with Daxx

Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in Kaposi's sarcoma (KS) lesion and play a key role in angiogenesis. Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) has multiple functions related to viral latency and KSHV-induced oncogenesis. In this report, we have identified Daxx as a LANA-binding protein by co-immunoprecipitation analysis of HeLa cells stably expressing LANA. LANA associated with Daxx in a PEL cell line infected with KSHV. LANA and Daxx also bound in vitro, suggesting direct interaction. From the results of binding assays, a region containing the Glu/Asp-rich domain within LANA, and a central region including the second paired amphipathic helix within Daxx contributed to the interaction. To address the physiological significance of this interaction, we focused on a Daxx-mediated VEGF receptor gene regulation. We found that Daxx repressed Ets-1-dependent Flt-1/VEGF receptor-1 gene expression, and that LANA inhibited the repression by Daxx in a reporter assay. Analyses of flow cytometry and real-time PCR revealed that expression of VEGF receptor-1 and -2 in LANA-expressing human umbilical vein endothelial cells (HUVECs) significantly increased. Co-immunoprecipitation and immunoblotting experiments suggested that LANA-bound Daxx to inhibit the interaction between Daxx and Ets-1. Chromatin immunoprecipitation assays showed that Daxx associated with VEGF receptor-1 promoter in HUVECs, and that LANA expression reduced this association. These results suggested that LANA contributes to a high expression of VEGF receptors in KS lesion by interfering with the interaction between Daxx and Ets-1.     

EC5S ubiquitin complex is recruited by KSHV latent antigen LANA for degradation of the VHL and p53 tumor suppressors

Cellular protein degradation pathways can be utilized by viruses to establish an environment that favors their propagation. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) directly functions as a component of the EC5S ubiquitin complex targeting the tumor suppressors von Hippel-Lindau (VHL) and p53 for degradation. We have characterized a suppressor of cytokine signaling box-like motif within LANA composed of an Elongin B and C box and a Cullin box, which is spatially located at its amino and carboxyl termini. This motif is necessary for LANA interaction with the Cul5-Elongin BC complex, to promote polyubiquitylation of cellular substrates VHL and p53 in vitro via its amino- and carboxyl-terminal binding domain, respectively. In transfected cells as well as KSHV-infected B lymphoma cells, LANA expression stimulates degradation of VHL and p53. Additionally, specific RNA interference-mediated LANA knockdown stabilized VHL and p53 in primary effusion lymphoma cells. Thus, manipulation of tumor suppressors by LANA potentially provides a favorable environment for progression of KSHV-infected tumor cells.     

Phosphorylation of the Chromatin Binding Domain of KSHV LANA

The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1–329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3–21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.     

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.     

The ubiquitin-specific protease USP7 modulates the replication of Kaposi's sarcoma-associated herpesvirus latent episomal DNA

Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.     

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.     

Ribosomal protein S6 interacts with the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus

The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA.     

Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen dysregulates expression of MCL-1 by targeting FBW7

Primary effusion lymphoma (PEL) is an aggressive B cell lymphoma that is etiologically linked to Kaposi’s sarcoma-associated herpesvirus (KSHV). Despite standard multi-chemotherapy treatment, PEL continues to cause high mortality. Thus, new strategies to control PEL are needed urgently. Here, we show that a phosphodegron motif within the KSHV protein, latency-associated nuclear antigen (LANA), specifically interacts with E3 ubiquitin ligase FBW7, thereby competitively inhibiting the binding of the anti-apoptotic protein MCL-1 to FBW7. Consequently, LANA-FBW7 interaction enhances the stability of MCL-1 by preventing its proteasome-mediated degradation, which inhibits caspase-3-mediated apoptosis in PEL cells. Importantly, MCL-1 inhibitors markedly suppress colony formation on soft agar and tumor growth of KSHV⁺PEL/BCBL-1 in a xenograft mouse model. These results strongly support the conclusion that high levels of MCL-1 expression enable the oncogenesis of PEL cells and thus, MCL-1 could be a potential drug target for KSHV-associated PEL. This work also unravels a mechanism by which an oncogenic virus perturbs a key component of the ubiquitination pathway to induce tumorigenesis.