ABSTRACT: NF-kB essential modulator (NEMO) is essential for KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13- induced gene expression and its N-terminal 251 resdidues are sufficent for this process
Project description:We report here that KSHV viral infection targets the NF-kB pathway which is crucial for cell survival. KSHV protein vFLIP K13 is known to directly interact with cellular protein NEMO of the NF-kB pathway. We used gene expression array to suggets that the interaction of K13 with NEMO is important to activate NF-kB pathway.
Project description:We report here that KSHV viral infection targets the NF-kB pathway which is crucial for cell survival. KSHV protein vFLIP K13 is known to directly interact with cellular protein NEMO of the NF-kB pathway. We used gene expression array to suggets that the interaction of K13 with NEMO is important to activate NF-kB pathway. We employed gene array analysis followed by bioinformatics analyses as well as biological pathways to determine the effect of K13 interaction with Nemo in Jurkat cell line system. Total RNA was isolated isolated using standard RNAeasy kit protocol (Qiagen). Their gene expression profiles were obtained using the Affymetrix Human Genome U133A 2.0 Array.
Project description:Integrated microarray and multiplex cytokine analyses of Kaposi's Sarcoma Asssociated Herpesvirus viral FLICE Inhibitory Protein K13 affected genes and cytokines in human blood vascular endothelial cells. The KSHV-encoded K13 protein is one of the few proteins to be expressed in latently-infected spindle cells and the ectopic expression of K13 in human vascular endothelial cells is sufficient to transform them into spindle cells. Experiment Overall Design: We have examined the effect of ectopic K13 expression on global gene expression in human umbilical vein endothelial cells (HUVEC).
Project description:Integrated microarray and multiplex cytokine analyses of Kaposi's Sarcoma Asssociated Herpesvirus viral FLICE Inhibitory Protein K13 affected genes and cytokines in human blood vascular endothelial cells. The KSHV-encoded K13 protein is one of the few proteins to be expressed in latently-infected spindle cells and the ectopic expression of K13 in human vascular endothelial cells is sufficient to transform them into spindle cells.
Project description:Host defense and inflammation are regulated by the NF-kB essential modulator (NEMO), a scaffolding protein with a broad immune cell and tissue expression profile. Hypomorphic mutations in inhibitor of nuclear factor kappa B kinase regulatory subunit gamma (IKBKG) encoding NEMO typically present with immunodeficiency. Here we characterized a novel pediatric autoinflammatory syndrome in 3 unrelated male patients with distinct X-linked IKBKG germ-line mutations that led to overexpression of a NEMO protein isoform lacking the domain encoded by exon 5 (NEMO-Dex5). This isoform failed to associate with TANK binding kinase 1 (TBK1), and dermal fibroblasts from affected patients activated NF-kB in response to TNF, but not TLR3 or RIG-I-like-receptor (RLR) stimulation when isoform levels were high. By contrast, T cells, monocytes and macrophages that expressed NEMO-Dex5 exhibited increased NF-kB activation and IFN production, and blood cells from these patients expressed a strong interferon and NF-kB transcriptional signature. Immune cells and TNF-stimulated dermal fibroblasts upregulated the inducible IKK protein (IKKi) that was stabilized by NEMO-Dex5, promoting type I IFN induction and antiviral responses. These data reveal how IKBKG mutations that lead to alternative splicing of skipping exon 5 cause a clinical phenotype we name NEMO Deleted exon 5 Autoinflammatory Syndrome (NDAS), distinct from the immunodeficiency syndrome resulting from loss-of-function IKBKG mutations.
Project description:Infection of Kaposi's sarcoma associated herpes virus (KSHV) has been linked to the development of primary effusion lymphoma (PEL), which is characterized by the loss of expression of B cell markers and effusions in the body cavities. This unique clinical feature of PEL has been attributed to their distinctive gene expression profile which shows overexpression of genes in various signaling pathways. KSHV-encoded latent protein vFLIP K13 has been shown to promote the survival and proliferation of PEL cells. In this study, we have employed gene array analysis followed by bioinformatics analysis of coordinated transcriptional factors network as well as biological pathways to characterize the effect of K13 on PEL-derived BCBL1 cells. We observed that genes associated with Cytokine signaling, Cell death, NF-kappaB and Cell adhesion pathways were differentially regulated by K13. We used computational approach complemented with microarrays to detail the global gene expression and identified distinct classes of differentially regulated genes in various cellular processes. We employed gene array analysis followed by bioinformatics analyses of coordinated transcriptional factors network as well as biological pathways to characterize the effect of K13 on in PEL-derived BCBL1 cells.
Project description:Infection of Kaposi's sarcoma associated herpes virus (KSHV) has been linked to the development of primary effusion lymphoma (PEL), which is characterized by the loss of expression of B cell markers and effusions in the body cavities. This unique clinical feature of PEL has been attributed to their distinctive gene expression profile which shows overexpression of genes in various signaling pathways. KSHV-encoded latent protein vFLIP K13 has been shown to promote the survival and proliferation of PEL cells. In this study, we have employed gene array analysis followed by bioinformatics analysis of coordinated transcriptional factors network as well as biological pathways to characterize the effect of K13 on PEL-derived BCBL1 cells. We observed that genes associated with Cytokine signaling, Cell death, NF-kappaB and Cell adhesion pathways were differentially regulated by K13. We used computational approach complemented with microarrays to detail the global gene expression and identified distinct classes of differentially regulated genes in various cellular processes.
Project description:Many immune responses depend upon activation of NF-κB, a key transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation) domains in N4BP1 mediate the interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated), but not TRIF-dependent (TLR3 or TLR4-mediated), NF-κB activation leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also exhibit diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.
