Clinical Manifestations of Kaposi Sarcoma Herpesvirus Lytic Activation: Multicentric Castleman Disease (KSHV–MCD) and the KSHV Inflammatory Cytokine Syndrome

Soon after the discovery of Kaposi sarcoma (KS)-associated herpesvirus (KSHV), it was appreciated that this virus was associated with most cases of multicentric Castleman disease (MCD) arising in patients infected with human immunodeficiency virus. It has subsequently been recognized that KSHV–MCD is a distinct entity from other forms of MCD. Like MCD that is unrelated to KSHV, the clinical presentation of KSHV–MCD is dominated by systemic inflammatory symptoms including fevers, cachexia, and laboratory abnormalities including cytopenias, hypoalbuminemia, hyponatremia, and elevated C-reactive protein. Pathologically KSHV–MCD is characterized by polyclonal, IgM-lambda restricted plasmacytoid cells in the intrafollicular areas of affected lymph nodes. A portion of these cells are infected with KSHV and a sizable subset of these cells express KSHV lytic genes including a viral homolog of interleukin-6 (vIL-6). Patients with KSHV–MCD generally have elevated KSHV viral loads in their peripheral blood. Production of vIL-6 and induction of human (h) IL-6 both contribute to symptoms, perhaps in combination with overproduction of IL-10 and other cytokines. Until recently, the prognosis of patients with KSHV–MCD was poor. Recent therapeutic advances targeting KSHV-infected B cells with the anti-CD20 monoclonal antibody rituximab and utilizing KSHV enzymes to target KSHV-infected cells have substantially improved patient outcomes. Recently another KSHV-associated condition, the KSHV inflammatory cytokine syndrome (KICS) has been described. Its clinical manifestations resemble those of KSHV–MCD but lymphadenopathy is not prominent and the pathologic nodal changes of KSHV–MCD are absent. Patients with KICS exhibit elevated KSHV viral loads and elevation of vIL-6, homolog of human interleukin-6 and IL-10 comparable to those seen in KSHV–MCD; the cellular origin of these is a matter of investigation. KICS may contribute to the inflammatory symptoms seen in some patients with severe KS or primary effusion lymphoma. Additional research is needed to better define the clinical spectrum of KICS and its relationship to KSHV–MCD. In additional, research is needed to better understand the pathogenesis and epidemiology of both KICS and KSHV–MCD, as well as the optimal therapy for both of these disorders

EBV-Encoded LMP1 Upregulates Igκ 3′Enhancer Activity and Igκ Expression in Nasopharyngeal Cancer Cells by Activating the Ets-1 through ERKs Signaling

Accumulating evidence indicates that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cells, express immunoglobulins (Igs). We previously found that the expression of the kappa light chain protein in NPC cells can be upregulated by the EBV-encoded latent membrane protein 1 (LMP1). In the present study, we used NPC cell lines as models and found that LMP1-augmented kappa production corresponds with elevations in ERKs phosphorylation. PD98059 attenuates LMP1-induced ERKs phosphorylation resulting in decreased expression of the kappa light chain. ERK-specific small interfering RNA blunts LMP1-induced kappa light chain gene expression. Luciferase reporter assays demonstrate that immunoglobulin κ 3′ enhancer (3′Eκ) is active in Igκ-expressing NPC cells and LMP1 upregulates the activity of 3′Eκ in NPC cells. Moreover, mutation analysis of the PU binding site in 3′Eκ and inhibition of the MEK/ERKs pathway by PD98059 indicate that the PU site is functional and LMP1-enhanced 3′Eκ activity is partly regulated by this site. PD98059 treatment also leads to a concentration-dependent inhibition of LMP1-induced Ets-1 expression and phosphorylation, which corresponds with a dose-dependent attenuation of LMP1-induced ERK phosphorylation and kappa light chain expression. Suppression of endogenous Ets-1 by small interfering RNA is accompanied by a decrease of Ig kappa light chain expression. Gel shift assays using nuclear extracts of NPC cells indicate that the transcription factor Ets-1 is recruited by LMP1 to the PU motif within 3′Eκ in vitro. ChIP assays further demonstrate Ets-1 binding to the PU motif of 3′Eκ in cells. These results suggest that LMP1 upregulates 3′Eκ activity and kappa gene expression by activating the Ets-1 transcription factor through the ERKs signaling pathway. Our studies provide evidence for a novel regulatory mechanism of kappa expression, by which virus-encoded proteins activate the kappa 3′ enhancer through activating transcription factors in non-B epithelial cancer cells

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms

The PU binding site is involved in LMP1-induced human 3′E_κ enhancer activity in NPC cells.

<p>(A) Schematic diagram of the human 3′E_κ core fragment-containing DNA portion used in these experiments. The position of the PU binding site is shown. For simplicity, other protein-binding sites in the 3′E_κ are not shown. The expansion of the PU binding site gives its wild-type sequence and the nucleotides replaced by mutations are underlined. Arrows indicate the nucleotides introduced by mutations. (B) Insertion sites for the DNA fragment in the <i>pGL3-β</i> vector, which contains the human <i>β-globin</i> promoter and the <i>luciferase</i> reporter gene. (C) Comparison of 3′E_κ activity in human nasopharyngeal carcinoma cell lines. The constructs carrying the wild-type PU sequence (<i>pβ-3′E_κwt</i>), mutant PU sequence (<i>pβ-3′E_κmt</i>), <i>pGL3-β</i> or <i>pGL3-Basic</i> with the internal control plasmid <i>pRL-SV40</i> were transiently co-transfected into HNE2 and HNE2-LMP1 cells. Luciferase reporter assays were performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032624#s2" target="_blank">Materials and methods</a>”. Values for firefly luciferase activity were normalized to those obtained for <i>Renilla</i> luciferase activity. Values obtained for cells transfected with <i>pβ-3′E_κwt, pβ-3′E_κmt</i> and <i>pGL3-β</i> were divided by the corresponding values obtained for cells transfected with <i>pGL3-Basic</i>. Data are shown as means ± S.D. of three independent experiments performed in triplicate. Statistical significance: #<i>p</i><0.05 <i>vs. pGL3-β</i>-transfected HNE2 cells; *<i>p</i><0.01 <i>vs. pGL3-β</i>-transfected HNE2-LMP1 cells; **<i>p</i><0.05 <i>vs. pβ-3′E_κw</i>t-transfected HNE2-LMP1 cells.</p

The MEK inhibitor, PD98059, abolishes the LMP1-increased 3′E_κ enhancer activity.

<p>HNE2 and HNE2-LMP1 cells were co-transfected with <i>pβ-3′E_κwt, pGL3-β,</i> or <i>pGL3-Basic</i> and the internal control <i>pRL-SV40</i> plasmids. Cells were incubated for 24 hr and then treated with PD98059 (50 µM) or DMSO (0.1%) for an additional 12 hr after which activity of firefly and <i>Renilla</i> luciferase was monitored as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032624#s2" target="_blank">Materials and Methods</a>”. Values for firefly luciferase activity were normalized to those obtained for <i>Renilla</i> luciferase activity. Values obtained for <i>pβ-3′E_κwt-</i> or <i>pGL3-β</i>-transfected cells were divided by the corresponding values obtained for <i>pGL3-Basic-</i>transfected cells. Data are shown as means ± S.D. of three independent experiments performed in triplicate. Statistical significance: *<i>p</i><0.05.</p

Inhibition of the ERKs signaling pathway blunts LMP1-increased kappa light chain expression at both the mRNA and protein levels.

<p>(A) HNE2-LMP1 cells were treated with the indicated concentrations of PD98059 or 0.1% DMSO for 2 hr. Whole cell lysates were prepared and total and phosphorylated ERKs levels were determined by Western blotting. (B) HNE2-LMP1 cells were treated with the indicated concentrations of PD98059 or 0.1% DMSO for 12 hr. Kappa light chain expression in NPC cells was assessed by Western blotting using a specific antibody. (C) HNE2 and HNE2-LMP1 cells were treated with 50 µM PD98059 or 0.1% DMSO for 12 hr and Western blotting was performed to detect kappa light chain expression. (D) HNE2 and HNE2-LMP1 cells were incubated with medium containing the indicated concentration of PD98059 or 0.1% DMSO for 12 hr. Total RNA was isolated from cells and subjected to RT-PCR, using specific primers designed to amplify <i>kappa light chain</i> and <i>actin</i> mRNAs. (E) HNE2-LMP1 cells were transfected with <i>si-ERK</i> or scrambled oligonucleotide. ERK and Ig kappa protein levels were detected by immunoblotting. The results shown are representative of three independent experiments. Phosphorylation or total expression level for each protein as well as mRNA was estimated by densitometry and are presented as a ratio to the respective loading control (right panels). XG7 and XG6 cells are shown as positive and negative controls, respectively, for kappa light chain.</p