Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat

BACKGROUND: The nuclear transcription factor NF-κB binds to the HIV-1 long terminal repeat (LTR) and is a key regulator of HIV-1 gene expression in cells latently infected with this virus. In this report, we have analyzed the ability of Kaposi's sarcoma associate herpes virus (KSHV, also known as Human Herpes virus 8)-encoded viral FLIP (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein) K13 to activate the HIV-1 LTR. RESULTS: We present evidence that vFLIP K13 activates HIV-1 LTR via the activation of the classical NF-κB pathway involving c-Rel, p65 and p50 subunits. K13-induced HIV-1 LTR transcriptional activation requires the cooperative interaction of all three components of the IKK complex and can be effectively blocked by inhibitors of the classical NF-κB pathway. K13 mutants that lacked the ability to activate the NF-κB pathway also failed to activate the HIV-1 LTR. K13 could effectively activate a HIV-1 LTR reporter construct lacking the Tat binding site but failed to activate a construct lacking the NF-κB binding sites. However, coexpression of HIV-1 Tat with K13 led to synergistic activation of HIV-1 LTR. Finally, K13 differentially activated HIV-1 LTRs derived from different strains of HIV-1, which correlated with their responsiveness to NF-κB pathway. CONCLUSIONS: Our results suggest that concomitant infection with KSHV/HHV8 may stimulate HIV-1 LTR via vFLIP K13-induced classical NF-κB pathway which cooperates with HIV-1 Tat protein

Kaposi’s Sarcoma Associated Herpesvirus Encoded Viral FLICE Inhibitory Protein K13 Activates NF-κB Pathway Independent of TRAF6, TAK1 and LUBAC

BACKGROUND: Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK) complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm), which have mutation in the Sharpin gene (Sharpin(cpdm/cpdm)). Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKβ, which resulted in their activation by "T Loop" phosphorylation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKβ and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies

A Computational Profiling of Changes in Gene Expression and Transcription Factors Induced by vFLIP K13 in Primary Effusion Lymphoma

Infection with Kaposi's sarcoma associated herpesvirus (KSHV) has been linked to the development of primary effusion lymphoma (PEL), a rare lymphoproliferative disorder that is characterized by loss of expression of most B cell markers and effusions in the body cavities. This unique clinical presentation of PEL has been attributed to their distinctive plasmablastic gene expression profile that shows overexpression of genes involved in inflammation, adhesion and invasion. KSHV-encoded latent protein vFLIP K13 has been previously shown to promote the survival and proliferation of PEL cells. In this study, we employed gene array analysis to characterize the effect of K13 on global gene expression in PEL-derived BCBL1 cells, which express negligible K13 endogenously. We demonstrate that K13 upregulates the expression of a number of NF-κB responsive genes involved in cytokine signaling, cell death, adhesion, inflammation and immune response, including two NF-κB subunits involved in the alternate NF-κB pathway, RELB and NFKB2. In contrast, CD19, a B cell marker, was one of the genes downregulated by K13. A comparison with K13-induced genes in human vascular endothelial cells revealed that although there was a considerable overlap among the genes induced by K13 in the two cell types, chemokines genes were preferentially induced in HUVEC with few exceptions, such as RANTES/CCL5, which was induced in both cell types. Functional studies confirmed that K13 activated the RANTES/CCL5 promoter through the NF-κB pathway. Taken collectively, our results suggest that K13 may contribute to the unique gene expression profile, immunophenotype and clinical presentation that are characteristics of KSHV-associated PEL

K13 blocks KSHV lytic replication and deregulates vIL6 nad hIL6 expression: A model of lytic replication induced clonal selection in viral oncogenesis

Background. Accumulating evidence suggests that dysregulated expression of lytic genes plays an important role in KSHV (Kaposi's sarcoma associated herpesvirus) tumorigenesis. However, the molecular events leading to the dysregulation of KSHV lytic gene expression program are incompletely understood. Methodoloxy/Principal Findings. We have studied the effect of KSHV-encoded latent protein vFLIP K13, a potent activator of the NF-κB pathway, on lytic reactivation of the virus. We demonstrate that K13 antagonizes RTA, the KSHV lytic-regulator, and effectively blocks the expression of lytic proteins, production of infectious virions and death of the infected cells. Induction of lytic replication selects for clones with increased K13 expression and NF-κB activity, while siRNA-mediated silencing of K13 induces the expression of lytic genes. However, the suppressive effect of K13 on RTA-induced lytic genes is not uniform and it falls to block RTA-induced viral IL6 secretion and cooperates with RTA to enhance cellular IL-6 production, thereby dysregulating the lytic gene expression program. Conclusions/Significance. Our results support a model in which ongoing KSHV, lytic replication selects for clones with progressively higher levels of K13 expression and NF-κB activity, which in turn drive KSHV tumorigenesis by not only directly stimulating cellular survival and proliferation, but also indirectly by dysregulating the viral lytic gene program and allowing non-lytic production of growth-promoting viral and cellular genes. Lytic Replication-Induced Clonal Selection (LyRICS) may represent a general mechanism in viral oncogenesis. 2007 Zhao et al

Transcription factors with binding sites over-represented in promoters of genes upregulated by K13 in BCBL1 and HUVECs.

<p>(Genes upregulated more than 1.5-fold were analyzed using the JASPAR and TRANSFEC databases. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037498#s3" target="_blank">Results</a> with a p-value of less than 0.05 are shown. % input refers to the number of gene promoters bearing the specific motif compared to total number screened.</p

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat-1

<p><b>Copyright information:</b></p><p>Taken from "Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat"</p><p>Retrovirology 2005;2():9-9.</p><p>Published online 15 Feb 2005</p><p>PMCID:PMC554086.</p><p>Copyright © 2005 Sun et al; licensee BioMed Central Ltd.</p>(75 ng/well) and an pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well) and luciferase reporter assay performed as described in Fig. 1A. The values shown are averages (mean ± SEM) of one representative experiment out of three in which each transfection was performed in duplicate. B. HIV-1 LTR activation by wild-type and mutant K13 constructs. The experiment was performed as described for Fig. 1A

Gene set enrichment analysis.

<p>For Gene set enrichment analysis of signatures genes from BCBL1-K13 (top panel) and HUVEC-K13 (lower panel), the t-test was graphed for each correlated gene in the ranked dataset. Three GSEA enrichment plots for representative biological pathways (Cytokine, NF-κB and Inflammatory) enriched in 4OHT-treated BCBL1-K13-ER^TAM and HUVEC-K13-ER^TAM are shown. The top portion of each GSEA plot shows the running enrichment score for validated genes specific for particular pathway as it moves down the ranked list. The bottom portion of each plot shows the value of ranking matrices as it moves down the list of ranked genes. The red horizontal bar which terminate with blue color indicate shift from positively correlated genes (red) to negatively correlated genes (blue). Further detailed interpretation about these plots can be found at Broad Institute web site (<a href="http://www.broadinstitute.org/gsea/index.jsp" target="_blank">http://www.broadinstitute.org/gsea/index.jsp</a>).</p

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat-0

<p><b>Copyright information:</b></p><p>Taken from "Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat"</p><p>Retrovirology 2005;2():9-9.</p><p>Published online 15 Feb 2005</p><p>PMCID:PMC554086.</p><p>Copyright © 2005 Sun et al; licensee BioMed Central Ltd.</p>onstruct (75 ng/well), and the experiment was performed as described under "Materials and Methods." The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. A dose-response analysis of HIV-1 LTR activation by K13 and pro-inflammatory cytokines. 293T cells were transfected with the indicated amounts of a K13 expression plasmid and luciferase assay performed 36 h post-transfection as described for (A). The total amount of transfected DNA was kept constant by adding an empty vector. For experiments involving TNF-α and IL-1β, cells were treated with the indicated concentration of cytokines 12 h after transfection of the reporter plasmids and assayed for reporter activity after 24 h of stimulation. C. K13 activates HIV-1 LTR in Cos-7 cells. The experiment was performed as described in 1A except LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA) was used for transfection and Renilla luciferase was used for normalization. D. K13 activates HIV-1 LTR in Jurkat cells. The experiment was performed as described for 1C by using LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA)

Validation of gene array data by qRT-PCR.

<p>(A) Twenty five genes from NF-κB, cytokine, and inflammatory pathways were randomly selected and their relative mRNA levels in mock and 4OHT-treated vector and K13-ER^TAM-expressing BCBL1 cells were examined using qRT-PCR. Real-time PCR reactions were performed in triplicate and the data presented as fold change mean ±S.E in target gene expression (*p<0.05; Student's t-test). (B) Pearson Correlation coefficient between gene expression array and real time PCR showed a significant agreement (Correlation coefficient 0.88; p<0.0001).</p