The ubiquitin E3 ligase RAUL negatively regulates type i interferon through ubiquitination of the transcription factors IRF7 and IRF3.

In the course of combating infectious agents, type I interferon (IFN) needs a timely downregulation mechanism to avoid detrimental overreaction. Here we showed a mechanism for restraining type I IFN responses, which relied on a HECT domain ubiquitin (Ub) E3 ligase, RAUL. RAUL limited type I IFN production by directly catalyzing lysine 48-linked polyubiquitination of both interferon regulatory factor 7 (IRF7) and IRF3 followed by proteasome-dependent degradation. Suppression of RAUL by dominant-negative RAUL or siRNA augmented both basal and virus-induced production of type I IFN, which resulted in reduced viral replication. The Kaposi's sarcoma-associated herpes virus immediate-early lytic cycle trigger protein RTA recruited this mechanism to augment its countermeasures against the host antiviral response. These results unveil a previously unrecognized "brake mechanism" for type I IFN that maintains proper low amounts of type I IFN under physiological conditions and restrains its magnitude when the antiviral response intensifies.

Upregulation of Id1 by Epstein-Barr virus-encoded LMP1 confers resistance to TGFbeta-mediated growth inhibition.

BACKGROUND: Epstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFbeta-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised. RESULTS: In this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFbeta-SMAD-mediated transcription and renders cells refractory to TGFbeta-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFbeta-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFbeta stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFbeta. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFbeta-induced Id1 upregulation. CONCLUSION: The current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFbeta-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformation.

IFN-producing killer dendritic cells are antigen-presenting cells endowed with T-cell cross-priming capacity.

IFN-producing killer dendritic cells (IKDC) represent a recently discovered cell type in the immune system that possesses a number of functions contributing to innate and adaptive immunity, including production of type 1 and 2 IFNs, interleukin (IL)-12, natural killing, and ultimately antigen presentation to naïve T cells. Here, we compared in vitro and in vivo responses of mouse IKDC, conventional dendritic cells (DC), and natural killer (NK) cells to murine cytomegalovirus infection and found distinct functions among these cell subsets. Upon recognition of infected fibroblasts, IKDC, as well as NK, produced high level of IFN-gamma, but unlike NK, IKDC simultaneously produced IL-12p40 and up-regulated MHC class II (MHC-II) and costimulatory molecules. Using MHC-II molecule expression as a phenotypic marker to distinguish activated IKDC from activated NK, we further showed that highly purified MHC-II(+) IKDC but not NK cross-present MHC class I-restricted antigens derived from MCMV-infected targets to CD8(+) T cells in vitro and in vivo. Our findings emphasize the unique nature of IKDC as a killer antigen-presenting cell directly linking innate and adaptive immunity.

Effects of chemotherapy in AIDS-associated non-Hodgkin's lymphoma on Kaposi's sarcoma herpesvirus DNA in blood.

PURPOSE: To determine the relative frequency with which Kaposi's sarcoma-associated herpesvirus/HHV-8 (KSHV) DNA is detected in peripheral-blood mononuclear cells (PBMCs) and in plasma of patients with AIDS-KS and AIDS-associated non-Hodgkin's lymphoma (NHL; AIDS-NHL); to determine whether the presence of viral DNA in plasma reflects lysis of tumor cells or reflects the presence of viremia (ie, virion-encapsidated DNA); and to determine the effect of lymphoma therapy on KSHV DNA. PATIENTS AND METHODS: Samples were obtained from patients enrolled in AIDS Malignancy Consortium clinical trials and from healthy donors. Real time PCR was used to quantify KSHV DNA in peripheral blood mononuclear cells (PBMC) and plasma. DNase digestion and fragment size determination studies were used to characterize the DNA detected. RESULTS: In patients with AIDS-KS, KSHV DNA was detected in PBMC (54%) and in plasma (62%). In patients with AIDS-NHL, KSHV DNA was detected in PBMC (19%) and in plasma (22%). Median copy numbers also differed. KSHV DNA in plasma appeared to be encapsidated. In six patients with AIDS-NHL who were treated with chemotherapy (with or without rituximab), KSHV copy number declined in PBMC and in plasma. CONCLUSION: KSHV DNA is sometimes detected in PBMC or in plasma of patients with AIDS-NHL without KS. Among patients with KSHV DNA detected in PBMC or in plasma, copy number does not distinguish between patients with AIDS-NHL and AIDS-KS. KSHV DNA in plasma likely reflects viremia and not simply lysis of tumor or other KSHV-infected cells. KSHV DNA copy number in PBMC and in plasma declined with lymphoma-directed cytotoxic chemotherapy in each of the six patients studied.

The KSHV immediate-early transcription factor RTA encodes ubiquitin E3 ligase activity that targets IRF7 for proteosome-mediated degradation.

Many viruses encode proteins that counteract the development of the interferon (IFN)-mediated antiviral state. Here, we report that interferon regulatory factor 7 (IRF7), a key mediator of type I IFN induction, is targeted for degradation by binding to the RTA immediate-early nuclear transcription factor encoded by Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8). Cotransfection with RTA blocked IRF7-mediated IFNalpha and IFNbeta mRNA production and promoted the ubiquitination and degradation of IRF7 protein in a proteasome-dependent fashion. Addition of RTA also promoted polyubiquitination of IRF7 in an in vitro cell free assay, demonstrating that RTA itself acts as a ubiquitin E3 ligase. RTA also autoregulated its own polyubiquitination and stability, and both activities were abolished by point mutations in a Cys plus His-rich N-terminal domain. Therefore, manipulation of the stability and function of IRF7 by the KSHV RTA transcription factor provides an unexpected regulatory strategy for circumventing the innate immune defence system.

Expression of herpes simplex virus type 2 protein ICP10 PK rescues neurons from apoptosis due to serum deprivation or genetic defects.

Previous studies have shown that the herpes simplex virus type 2 protein kinase ICP10 PK activates the Ras/MEK/MAPK pathway in nonneuronal cells. Here we report that ectopically expressed ICP10 PK has anti-apoptotic activity in various paradigms of neuronal cell death. Neuronally differentiated PC12 cells and primary murine hippocampal cultures transfected with an expression vector for ICP10 PK were protected from cell death resulting from growth factor withdrawal. Protection from apoptosis was also seen in ICP10 PK-transfected hippocampal neurons from the trisomy 16 mouse, a naturally occurring genetic abnormality the human analog of which is Down syndrome. Cells transfected with an expression vector for a mutant that lacks kinase activity were not protected, although it was expressed as well as ICP10 PK. The data indicate that ICP10 PK has a broad anti-apoptotic activity in neuronal cells which depends on a functional PK.

Rescue of synthetic salmonid rhabdovirus minigenomes.

Synthetic T7-driven cDNA minigenomes containing the bacterial chloramphenicol acetyltransferase gene as a reporter were derived from the genome of two salmonid novirhabdoviruses, infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). We showed that an exogenous IHNV RNA minigenome transfected into fish cells could be rescued following IHNV infection as it was replicated, encapsidated and transcribed. When cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3), transfected with the plasmid carrying the IHNV minigenome (genomic- and antigenomic-sense) and superinfected with IHNV, rescue of the minigenome was more efficient. Heterologous VHSV/IHNV rescue experiments failed. Finally, when the IHNV N, P and L proteins were expressed from cDNAs in cells, the minigenome was also successfully rescued, indicating that the nucleocapsid proteins were biologically functional. These data represent the first example of rescue experiments for non-mammalian rhabdoviruses replicating at a low temperature.

CCAAT/enhancer-binding protein-alpha is induced during the early stages of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic cycle reactivation and together with the KSHV replication and transcription activator (RTA) cooperatively stimulates the viral RTA, MTA, and PAN promoters.

During the immediate-early (IE) phase of reactivation from latency, the Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator protein (RTA) (or ORF50) is thought to be the most critical trigger that upregulates expression of many downstream viral lytic cycle genes, including the delayed-early (DE) gene encoding the replication-associated protein (RAP) (or K8). RAP physically interacts with and stabilizes the cellular transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha), leading to upregulated expression of the cellular C/EBPalpha and p21(CIP-1) proteins followed by G(0)/G(1) cell cycle arrest. Furthermore, RTA also interacts with C/EBPalpha, and both RAP and RTA cooperate with C/EBPalpha to activate the RAP promoter through binding to a strong proximal C/EBP binding site that also serves as an RTA-responsive element (RRE). Here we show that C/EBPalpha also activates the IE RTA promoter in transient-cotransfection reporter gene assays and that addition of either RTA or RAP enhances the effect. Electrophoretic mobility shift assay and deletion analysis revealed three C/EBP binding sites that mediate cooperative transactivation of the RTA promoter by C/EBPalpha and RTA. Furthermore, chromatin immunoprecipitation assay results showed that the endogenous C/EBPalpha, RTA, and RAP proteins all associate with RTA promoter sequences in tetradecanoyl phorbol acetate-induced primary effusion lymphoma (PEL) cells. Induction of endogenous KSHV RTA mRNA in PEL cells by exogenously introduced C/EBPalpha was confirmed by reverse transcription-PCR analysis and by double-label indirect immunofluorescence assays. Reciprocally, expression of exogenous RTA also led to an increase of endogenous C/EBPalpha expression that could be detected by Western immunoblot assays even in KSHV-negative DG75 cells. Cotransfected RTA also increased positive C/EBPalpha autoregulation of the C/EBPalpha promoter in transient-cotransfection reporter gene assays. Finally, C/EBPalpha proved to strongly activate the promoters of two other KSHV DE genes encoding PAN (polyadenylated nuclear) RNA and MTA (ORF57), which was again mediated by C/EBP binding sites that also contribute to RTA activation. Overall, these results support a model in which the cellular transcription factor C/EBPalpha and RTA:C/EBPalpha interactions play important roles both upstream and downstream of the two major KSHV regulatory proteins RTA and RAP during the early stages of lytic cycle reactivation.

Altered patterns of cellular gene expression in dermal microvascular endothelial cells infected with Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; also called human herpesvirus 8) is believed to be the etiologic agent of Kaposi's sarcoma, multicentric Castleman's disease, and AIDS-associated primary effusion lymphoma. KSHV infection of human dermal microvascular endothelial cells (DMVEC) in culture results in the conversion of cobblestone-shaped cells to spindle-shaped cells, a characteristic morphological feature of cells in KS lesions. All spindle-shaped cells in KSHV-infected DMVEC cultures express the latency-associated nuclear protein LANA1, and a subfraction of these cells undergo spontaneous lytic cycle induction that can be enhanced by tetradecanoyl phorbol acetate (TPA) treatment. To study the cellular response to infection by KSHV, we used two different gene array screening systems to examine the expression profile of either 2,350 or 9,180 human genes in infected compared to uninfected DMVEC cultures in both the presence and absence of TPA. In both cases, between 1.4 and 2.5% of the genes tested were found to be significantly upregulated or downregulated. Further analysis by both standard and real-time reverse transcription-PCR procedures directly confirmed these results for 14 of the most highly upregulated and 13 of the most highly downregulated genes out of a total of 37 that were selected for testing. These included strong upregulation of interferon-responsive genes such as interferon response factor 7 (IRF7) and myxovirus resistance protein R1, plus upregulation of exodus 2 beta-chemokine, RDC1 alpha-chemokine receptor, and transforming growth factor beta3, together with strong downregulation of cell adhesion factors alpha(4)-integrin and fibronectin plus downregulation of bone morphogenesis protein 4, matrix metalloproteinase 2, endothelial plasminogen activator inhibitor 1, connective tissue growth factor, and interleukin-8. Significant dysregulation of several other cytokine-related genes or receptors, as well as endothelial cell and macrophage markers, and various other genes associated with angiogenesis or transformation was also detected. Western immunoblot and immunohistochemical analyses confirmed that the cellular IRF7 protein levels were strongly upregulated during the early lytic cycle both in KSHV-infected DMVEC and in the body cavity-based lymphoma BCBL1 PEL cell line.