The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.     

Kaposi's sarcoma-associated herpesvirus (KSHV) oncoprotein K13 bypasses TRAFs and directly interacts with the IkappaB kinase complex to selectively activate NF-kappaB without JNK activation

Kaposi's sarcoma herpesvirus oncoprotein vFLIP K13 is a potent activator of NF-kappaB and plays a key role in viral pathogenesis. K13 contains a putative TRAF-interacting motif, which is reportedly required for its interaction with TRAF2. The K13-TRAF2 interaction is believed to be essential for the recruitment of K13 to the I-kappaB kinase (IKK) complex and for K13-induced NF-kappaB and JNK activation. In addition, TRAF3 has been reported to be required for K13-induced NF-kappaB and JNK activation. We have re-examined the role of the TRAFs in K13 signaling and report that mutations in the putative TRAF-interacting motif of K13 have no deleterious effect on its ability to interact with the IKK complex or activation of the NF-kappaB pathway. Furthermore, endogenously expressed TRAF2 and TRAF3 do not interact with K13 and play no role in K13-induced NF-kappaB activation or its interaction with the IKK complex. Finally, K13 does not activate the JNK pathway. Our results support a model in which K13 bypasses the upstream components of the tumor necrosis factor receptor signaling pathway and directly interacts with the IKK complex to selectively activate the NF-kappaB pathway without affecting the JNK pathway. Selective NF-kappaB activation by K13 might represent a novel strategy employed by the virus to promote latency.     

Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-κB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-κB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.     

Kaposi's sarcoma-associated herpesvirus latent gene vFLIP inhibits viral lytic replication through NF-kappaB-mediated suppression of the AP-1 pathway: a novel mechanism of virus control of latency

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is central to the evasion of host immune surveillances and induction of KSHV-related malignancies. The mechanism of KSHV latency remains unclear. Here, we show that the KSHV latent gene vFLIP promotes viral latency by inhibiting viral lytic replication. vFLIP suppresses the AP-1 pathway, which is essential for KSHV lytic replication, by activating the NF-kappaB pathway. Thus, by manipulating two convergent cellular pathways, vFLIP regulates both cell survival and KSHV lytic replication to promote viral latency. These results also indicate that the effect of the NF-kappaB pathway on KSHV replication is determined by the status of the AP-1 pathway and hence provide a mechanistic explanation for the contradictory role of the NF-kappaB pathway in KSHV replication. Since the NF-kappaB pathway is commonly activated during infection of gammaherpesviruses, these findings might have general implications for the control of gammaherpesviral latency.     

LMP1 strain variants: biological and molecular properties

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced transformation of B lymphocytes and is able to transform Rat-1 fibroblasts. LMP1 can activate a wide array of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt and NF-kappaB. Six sequence variants of LMP1, termed Alaskan, China 1, China 2, Med+, Med-, and NC, have been identified, and individuals can be infected with multiple variants. The frequencies of detection of these variants differ for various EBV-associated malignancies from different geographic regions. In this study, the biological and signaling properties of the LMP1 variants have been characterized. All of the LMP1 variants transformed Rat-1 fibroblasts, induced increased motility of HFK cells, and induced increased homotypic adhesion of BJAB cells. While all the variants activated the PI3K-Akt signaling pathway to similar extents, the Alaskan, China 1, and Med+ variants had limited binding to the E3 ubiquitin ligase component homologue of Slimb and had slightly enhanced NF-kappaB signaling. These findings indicate that the signature amino acid changes of the LMP1 variants do not hinder or enhance their in vitro transforming potentials or affect their signaling properties.     

Viral FLICE inhibitory protein of rhesus monkey rhadinovirus inhibits apoptosis by enhancing autophagosome formation

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to human herpesvirus 8 (HHV8). RRV encodes viral FLICE inhibitory protein (vFLIP), which has death effector domains. Little is known about RRV vFLIP. This study intended to examine its function in apoptosis. Here we found that RRV vFLIP inhibits apoptosis induced by tumor necrosis factor-α (TNF-α) and cycloheximide. In HeLa cells with vFLIP expression, the cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than those in controls. Cell viability of HeLa cells with vFLIP expression was significantly higher than control cells after apoptosis induction. However, RRV vFLIP appears unable to induce NF-κB signaling when tested in NF-κB reporter assay. RRV vFLIP was able to enhance cell survival under starved conditions or apoptosis induction. At early time points after apoptosis induction, autophagosome formation was enhanced and LC3-II level was elevated in cells with vFLIP and, when autophagy was blocked with chemical inhibitors, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis by enhancing autophagy to maintain cell survival. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These results indicate that vFLIP protects cells against apoptosis by enhancing autophagosome formation to extend cell survival. The finding of vFLIP's inhibition of apoptosis via the autophagy pathway provides insights of vFLIP in RRV pathogenesis.     

Herpesvirus saimiri vFLIP provides an antiapoptotic function but is not essential for viral replication, transformation, or pathogenicity

Apoptosis of infected cells is an important host defense mechanism, and many viruses have exploited antiapoptotic proteins that interfere with crucial cellular pathways. Viral FLICE inhibitory proteins (vFLIPs) are encoded by rhadinoviruses like herpesvirus saimiri, the related Kaposi's sarcoma-associated herpesvirus-human herpesvirus 8 (KSHV/HHV8), and the poxvirus responsible for molluscum contagiosum. The vFLIPs can block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8), and this prevents the initiation of the downstream caspase cascade. KSHV/HHV8 vFLIP overexpression can confer resistance to T-cell-mediated apoptosis and acts as a tumor progression factor in a murine B-cell lymphoma model. To analyze the function of herpesvirus vFLIPs in the genetic background of the virus and in a model for viral pathogenesis, we deleted the vFLIP gene (open reading frame 71) from the genome of herpesvirus saimiri strain C488. The viral deletion mutant was viable and replicated like the wild-type virus. An antiapoptotic effect could be attributed to the vFLIP gene, but we also show that the vFLIP gene of herpesvirus saimiri is dispensable for viral transformation of T cells in vitro and for pathogenicity in cottontop tamarins in vivo.     

Evaluation of the lytic origins of replication of Kaposi's sarcoma-associated virus/human herpesvirus 8 in the context of the viral genome

The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-DeltaOri-R, BAC36-DeltaOri-L, and BAC36-DeltaOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.     

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.     

Gene Regulation and Functional Alterations Induced by Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORFK13/vFLIP in Endothelial Cells

Kaposi''s sarcoma (KS) is an angioproliferative inflammatory disorder induced by endothelial cell infection with the KS-associated herpesvirus (KSHV). ORFK13/vFLIP, one of the KSHV genes expressed in KS, encodes a 188-amino-acid protein which binds to the Iκb kinase (IKK) complex to activate NF-κB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. Retroviral transduction of ORFK13/vFLIP into primary human endothelial cells induces the spindle morphology distinctive of KS cells and promotes the formation of abnormal vascular networks typical of KS vasculature; upregulates the expression of proinflammatory cytokines, chemokines, and interferon-responsive genes; and stimulates the adhesion of inflammatory cells characteristic of KS lesions. Thymidine phosphorylase, a cellular enzyme markedly induced by ORFK13/vFLIP, can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU), a potent thymidine synthase inhibitor, which blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1 to 1 μM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies.Kaposi''s sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is the etiological agent of Kaposi''s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman''s diseases. KS typically presents as a multicentric angioproliferative tumor characterized by multiple nodular or macular lesions often on the skin, and less frequently in the gastrointestinal tract and the lung. Histologically, the lesions consist of spindle cells infected with KSHV, inflammatory infiltrates of monocytes/macrophages, lymphocytes and other cells, and “vascular slits” replete of red blood cells (8). KS spindle cells are likely to be of endothelial lineage (19).In KS tissues, KSHV establishes a mostly latent infection characterized by expression of a limited number of viral genes that are likely important to the disease pathogenesis (30). ORFK13 is one such KSHV latent gene. Its gene product, called vFLIP (for viral Flice-like inhibitory protein) or K-FLIP, comprises two tandem death-effector domains that are often found in apoptotic signaling mediators such as cellular FLICE inhibitory protein (cFLIP) and caspase-8/FLICE. Consistent with its sequence similarity with cFLIP, vFLIP was found to inhibit caspase activation and prevent apoptotic cell death (39). Silencing ORFK13/vFLIP expression by RNA interference stopped PEL growth in vitro and in vivo, providing evidence of the essential role of K13/vFLIP in PEL pathogenesis (16). Transgenic mice of K13/vFLIP in lymphoid cells developed more lymphomas than controls (11). Similar to the viral proteins of many other lymphogenic viruses, K13/vFLIP activates NF-κB (1, 10, 25, 27, 42). By activating NF-κB and inhibiting the AP-1 pathway, K13/vFLIP was recently reported to promote viral latency (49).Recent studies have characterized selected effects of K13/vFLIP expression in primary endothelial cells transduced with ORFK13/vFLIP (15, 20, 29), providing important insights into its function. Here, we broadly investigated K13/vFLIP function in endothelial cells. By establishing stable retrovirus-mediated transduction of ORFK13/vFLIP in primary human endothelial cells, we have extensively characterized the biochemical and functional consequences of K13/vFLIP expression in these cells.     

The antiangiogenic factor 16K human prolactin induces caspase-dependent apoptosis by a mechanism that requires activation of nuclear factor-kappaB

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.     

Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1

The human enteric flora plays a significant role in intestinal health and disease. Certain enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination. IkappaB-alpha ubiquitination is catalyzed by the E3-SCF(betaTrCP) ubiquitin ligase, which is itself regulated via covalent modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Neddylation is a biochemical event associated with diverse cellular processes related to cell signaling, however, physiological regulation of cullin neddylation has not been described in mammalian systems. We report that interaction of nonpathogenic bacteria with epithelial cells resulted in a rapid loss of neddylated Cul-1 and consequent repression of the NF-kappaB pathway. This observation may explain the ability of intestinal bacterial communities to influence diverse eukaryotic processes in general and inflammatory tolerance of the mammalian intestinal epithelia specifically.     

Heparin-binding epidermal growth factor-like growth factor inhibits cytokine-induced NF-kappa B activation and nitric oxide production via activation of the phosphatidylinositol 3-kinase pathway

NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-kappaB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-kappaB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by approximately 2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-kappaB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-kappaB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-kappaB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.     

Viral FLIP impairs survival of activated T cells and generation of CD8+ T cell memory

Viral FLIPs (vFLIPs) interfere with apoptosis signaling by death-domain-containing receptors in the TNFR superfamily (death receptors). In this study, we show that T cell-specific transgenic expression of MC159-vFLIP from the human Molluscum contagiosum virus blocks CD95-induced apoptosis in thymocytes and peripheral T cells, but also impairs postactivation survival of in vitro activated primary T cells despite normal early activation parameters. MC159 vFLIP impairs T cell development to a lesser extent than does Fas-associated death domain protein deficiency or another viral FLIP, E8. In the periphery, vFLIP expression leads to a specific deficit of functional memory CD8(+) T cells. After immunization with a protein Ag, Ag-specific CD8(+) T cells initially proliferate, but quickly disappear and fail to produce Ag-specific memory CD8(+) T cells. Viral FLIP transgenic mice exhibit impaired CD8(+) T cell responses to lymphocytic choriomeningitis virus and Trypanosoma cruzi infections, and a specific defect in CD8(+) T cell recall responses to influenza virus was seen. These results suggest that vFLIP expression in T cells blocks signals necessary for the sustained survival of CD8(+) T cells and the generation of CD8(+) T cell memory. Through this mechanism, vFLIP proteins expressed by T cell tropic viruses may impair the CD8(+) T cell immune responses directed against them.     

Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation

Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-alpha (TGF-alpha) suggests involvement of TGF-alpha in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-alpha in tumorigenicity, we introduced a human TGF-alpha cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-1) cells. Synthesis and secretion of human TGF-alpha by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-alpha monoclonal antibodies prevent TGF-alpha expressing Rat-1 cells from forming colonies in soft agar.