Freshly isolated, resting natural killer (NK) cells are generally less lytic against target cells than in vitro interleukin 2 (IL-2)-activated NK cells. To investigate the basis for this difference, the contribution of several receptors to activation of human NK cells was examined. Target-cell lysis by IL-2-activated NK cells in a redirected, antibody-dependent cytotoxicity assay was triggered by a number of receptors. In contrast, cytotoxicity by resting NK cells was induced only by CD16, and not by NKp46, NKG2D, 2B4 (CD244), DNAM-1 (CD226), or CD2. Calcium flux in resting NK cells was induced with antibodies to CD16 and, to a weaker extent, antibodies to NKp46 and 2B4. Although NKp46 did not enhance CD16-mediated calcium flux, it synergized with all other receptors. 2B4 synergized with <em>3</em> other receptors, NKG2D and DNAM-1 each synergized with 2 other receptors, and CD2 synergized with NKp46 only. Resting NK cells were induced to secrete tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) and <em>interferon</em> gamma (IFN-gamma), and to kill target cells by engagement of specific, pair-wise combinations of receptors. Therefore, natural cytotoxicity by resting NK cells is induced only by mutual costimulation of nonactivating receptors. These results reveal distinct and specific patterns of synergy among receptors on resting NK cells.

The receptors for <em>interferon</em>-<em>alpha</em>/beta (IFN-<em>alpha</em>/beta) and IFN-gamma activate components of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, leading to the formation of at least two transcription factor complexes. STAT1 interacts with STAT2 and p48/IRF-9 to form the transcription factor IFN-stimulated gene factor <em>3</em> (ISGF<em>3</em>). STAT1 dimers form gamma-activated factor (GAF). ISGF<em>3</em> is induced mainly by IFN-<em>alpha</em>/beta, and GAF by IFN-gamma, although both factors can be activated by both types of IFN. Individuals with mutations in either chain of the IFN-gamma receptor (IFN-gammaR) are susceptible to infection with mycobacteria. A heterozygous STAT1 mutation that impairs GAF but not ISGF<em>3</em> activation has been found in other individuals with mycobacterial disease. No individuals with deleterious mutations in the IFN-<em>alpha</em>/beta signaling pathway have been described. We report here two unrelated infants homozygous with respect to mutated STAT1 alleles. Neither IFN-<em>alpha</em>/beta nor IFN-gamma activated STAT1-containing transcription factors. Like individuals with IFN-gammaR deficiency, both infants suffered from mycobacterial disease, but unlike individuals with IFN-gammaR deficiency, both died of viral disease. Viral multiplication was not inhibited by recombinant IFN-<em>alpha</em>/beta in cell lines from the two individuals. Inherited impairment of the STAT1-dependent response to human IFN-<em>alpha</em>/beta thus results in susceptibility to viral disease.

OBJECTIVE

Bevacizumab is an antibody that binds vascular endothelial growth factor and has activity in metastatic renal cell carcinoma (RCC). Interferon alfa (IFN-alpha) is the historic standard initial treatment for RCC. A prospective, randomized, phase III trial of bevacizumab plus IFN-alpha versus IFN-alpha monotherapy was conducted.

METHODS

Patients with previously untreated, metastatic clear cell RCC were randomly assigned to receive either bevacizumab (10 mg/kg intravenously every 2 weeks) plus IFN-alpha (9 million units subcutaneously three times weekly) or the same dose and schedule of IFN-alpha monotherapy in a multicenter phase III trial. The primary end point was overall survival (OS). Secondary end points were progression-free survival (PFS), objective response rate, and safety.

RESULTS

Seven hundred thirty-two patients were enrolled. The median OS time was 18.3 months (95% CI, 16.5 to 22.5 months) for bevacizumab plus IFN-alpha and 17.4 months (95% CI, 14.4 to 20.0 months) for IFN-alpha monotherapy (unstratified log-rank P = .097). Adjusting on stratification factors, the hazard ratio was 0.86 (95% CI, 0.73 to 1.01; stratified log-rank P = .069) favoring bevacizumab plus IFN-alpha. There was significantly more grade 3 to 4 hypertension (HTN), anorexia, fatigue, and proteinuria for bevacizumab plus IFN-alpha. Patients who developed HTN on bevacizumab plus IFN-alpha had a significantly improved PFS and OS versus patients without HTN.

CONCLUSIONS

OS favored the bevacizumab plus IFN-alpha arm but did not meet the predefined criteria for significance. HTN may be a biomarker of outcome with bevacizumab plus IFN-alpha.

Stimulation of naïve CD8+ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin-12 (IL-12) or <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>). CD4+ T cells condition dendritic cells (DCs) to effectively present antigen to CD8+ T cells, and this conditioning involves, at least in part, CD40-dependent upregulation of the production of these signal <em>3</em> cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation-induced non-responsiveness (AINR), a form of split anergy characterized by an inability to produce IL-2 to support continued expansion. If antigen remains present, IL-2 provided by CD4+ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL-2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8+ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.

This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-<em>alpha</em> gene transcription in peritoneal exudate macrophages was accompanied by induction of DNA-binding proteins that recognized with different affinities four elements related to the kappa B consensus motif and a Y-box motif. We suggest that the basal level of TNF-<em>alpha</em> expression in macrophages is due to the binding of a constitutive form of NF-kappa B, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the kappa B motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-kappa B, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. <em>3</em>:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-<em>alpha</em> transcription, diminished the basal level of nuclear NF-kappa B. Stimulation of Stimulation of TNF-<em>alpha</em> transcription in macrophages by lipopolysaccharide, gamma <em>interferon</em>, or cycloheximide led to an increased content of nuclear NF-kappa B. This induced factor represents a different form of NF-kappa B, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-kappa B contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-kappa B to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-kappa B was indeed detectable in the cytosol after deoxycholate treatment. UV cross-linking experiments and gel retardation assays indicated that the inducible form of NF-kappa B is in a higher-order complex with other proteins.

The <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>)-stimulated gene factor <em>3</em> (ISGF<em>3</em>), a transcriptional activator, contains three proteins, termed ISGF<em>3</em> <em>alpha</em> proteins, that reside in the cell cytoplasm until they are activated in response to IFN-<em>alpha</em>. Treatment of cells with IFN-<em>alpha</em> caused these three proteins to be phosphorylated on tyrosine and to translocate to the cell nucleus where they stimulate transcription through binding to IFN-<em>alpha</em>-stimulated response elements in DNA. IFN-gamma, which activates transcription through a different receptor and different DNA binding sites, also caused tyrosine phosphorylation of one of these proteins. The ISGF<em>3</em> <em>alpha</em> proteins may be substrates for one or more kinases activated by ligand binding to the cell surface and may link occupation of a specific polypeptide receptor with activation of transcription of a set of specific genes.

Influenza A is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans. The host innate immune Toll-like receptor (TLR) <em>3</em> was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded RNA (dsRNA). Thus, dsRNA may be critical for the outcome of the infection. Here we first compared the activation triggered by either influenza A virus or dsRNA in pulmonary epithelial cells. We established that TLR<em>3</em> is constitutively expressed in human alveolar and bronchial epithelial cells, and we describe its intracellular localization. Expression of TLR<em>3</em> was positively regulated by the influenza A virus and by dsRNA but not by other inflammatory mediators, including bacterial lipopolysaccharide, the cytokines tumor necrosis factor-<em>alpha</em> and interleukin (IL)-1beta, and the protein kinase C activator phorbol 12-myristate 1<em>3</em>-acetate. We also demonstrated that TLR<em>3</em> contributes directly to the immune response of respiratory epithelial cells to influenza A virus and dsRNA, and we propose a molecular mechanism by which these stimuli induce epithelial cell activation. This model involves mitogen-activated protein kinases, phosphatidylinositol <em>3</em>-kinase/Akt signaling, and the TLR<em>3</em>-associated adaptor molecule TRIF but not MyD88-dependent activation of the transcription factors NF-kappaB or <em>interferon</em> regulatory factor/<em>interferon</em>-sensitive response-element pathways. Ultimately, this signal transduction elicits an epithelial response that includes the secretion of the cytokines IL-8, IL-6, RANTES (regulated on activation normal T cell expressed and secreted), and <em>interferon</em>-beta and the up-regulation of the major adhesion molecule ICAM-1.

BACKGROUND

An effective vaccine for malaria is urgently needed. Naturally acquired immunity to malaria develops slowly, and induction of protection in humans can be achieved artificially by the inoculation of radiation-attenuated sporozoites by means of more than 1000 infective mosquito bites.

METHODS

We exposed 15 healthy volunteers--with 10 assigned to a vaccine group and 5 assigned to a control group--to bites of mosquitoes once a month for <em>3</em> months while they were receiving a prophylactic regimen of chloroquine. The vaccine group was exposed to mosquitoes that were infected with Plasmodium falciparum, and the control group was exposed to mosquitoes that were not infected with the malaria parasite. One month after the discontinuation of chloroquine, protection was assessed by homologous challenge with five mosquitoes infected with P. falciparum. We assessed humoral and cellular responses before vaccination and before the challenge to investigate correlates of protection.

RESULTS

All 10 subjects in the vaccine group were protected against a malaria challenge with the infected mosquitoes. In contrast, patent parasitemia (i.e., parasites found in the blood on microscopical examination) developed in all five control subjects. Adverse events were mainly reported by vaccinees after the first immunization and by control subjects after the challenge; no serious adverse events occurred. In this model, we identified the induction of parasite-specific pluripotent effector memory T cells producing interferon-gamma, tumor necrosis factor alpha, and interleukin-2 as a promising immunologic marker of protection.

CONCLUSIONS

Protection against a homologous malaria challenge can be induced by the inoculation of intact sporozoites. (ClinicalTrials.gov number, NCT00442<em>3</em>77.)

OBJECTIVE

The probiotic compound, VSL#<em>3</em>, is efficacious as maintenance therapy in pouchitis and ulcerative colitis. The aim of this study was to determine the efficacy of VSL#<em>3</em> as a primary therapy in the treatment of colitis in the interleukin (IL)-10 gene-deficient mouse. Mechanisms of action of VSL#<em>3</em> were investigated in T(84) monolayers.

METHODS

IL-10 gene-deficient and control mice received 2.8 x 10(8) colony-forming units per day of VSL#<em>3</em> for 4 weeks. Colons were removed and analyzed for cytokine production, epithelial barrier function, and inflammation. VSL#<em>3</em> or conditioned media was applied directly to T(84) monolayers.

RESULTS

Treatment of IL-10 gene-deficient mice with VSL#<em>3</em> resulted in normalization of colonic physiologic function and barrier integrity in conjunction with a reduction in mucosal secretion of tumor necrosis factor alpha and interferon gamma and an improvement in histologic disease. In vitro studies showed that epithelial barrier function and resistance to Salmonella invasion could be enhanced by exposure to a proteinaceous soluble factor secreted by the bacteria found in the VSL#<em>3</em> compound.

CONCLUSIONS

Oral administration of VSL#<em>3</em> was effective as primary therapy in IL-10 gene-deficient mice, and had a direct effect on epithelial barrier function.

The ribonucleoside analog ribavirin (1-beta-D-ribofuranosyl-1,2, 4-triazole-<em>3</em>-carboxamide) shows antiviral activity against a variety of RNA viruses and is used in combination with <em>interferon</em>-<em>alpha</em> to treat hepatitis C virus infection. Here we show in vitro use of ribavirin triphosphate by a model viral RNA polymerase, poliovirus <em>3</em>Dpol. Ribavirin incorporation is mutagenic, as it templates incorporation of cytidine and uridine with equal efficiency. Ribavirin reduces infectious poliovirus production to as little as 0. 00001% in cell culture. The antiviral activity of ribavirin correlates directly with its mutagenic activity. These data indicate that ribavirin forces the virus into 'error catastrophe'. Thus, mutagenic ribonucleosides may represent an important class of anti-RNA virus agents.

Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells. These RNAs activate signalling cascades that activate the genes that encode <em>alpha</em>- and beta-<em>interferon</em> (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs. RIG-I and MDA5 interact with another CARD protein, <em>interferon</em>-beta promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and nuclear factor (NF)-kappaB to the IFN-beta gene. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L, by 2',5'-linked oligoadenylate (2-5A) produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-beta expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved <em>3</em>' of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-beta expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-beta during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-beta in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential role in the innate antiviral immune response that relieves the requirement for direct sensing of non-self RNA.

Macrophages are supposed to play a key role in inflammatory and tumor angiogenesis. Their importance derives from (1) their ubiquitous presence in normal and especially inflamed tissues, (2) their potential to become activated in response to appropriate stimuli, and (<em>3</em>) their repertoire of secretory products. By release of proteases, growth factors (bFGF, GM-CSF, TGF-<em>alpha</em>, IGF-I, PDGF, VEGF/VPF, TGF-beta), and other monokines (IL-1, IL-6, IL-8, TNF-<em>alpha</em>, substance P, prostaglandins, <em>interferons</em>, thrombospondin 1), activated macrophages have the capability to influence each phase of the angiogenic process, such as alterations of the local extracellular matrix, induction of endothelial cells to migrate or proliferate, and inhibition of vascular growth with formation of differentiated capillaries. This review describes macrophage physiology and the influence of macrophage secretory products on the different phases of angiogenesis in vitro and in vivo.

<em>Interferon</em>-gamma (IFN-gamma) induces the transcription of the gene encoding a guanylate binding protein by activating a latent cytoplasmic factor, GAF (gamma-activated factor). GAF is translocated to the nucleus and binds a DNA element, the gamma-activated site. Through cross-linking and the use of specific antibodies GAF was found to be a 91-kilodalton DNA binding protein that was previously identified as one of four proteins in <em>interferon</em>-stimulated gene factor-<em>3</em> (ISGF-<em>3</em>), a transcription complex activated by IFN-<em>alpha</em>. The IFN-gamma-dependent activation of the 91-kilodalton DNA binding protein required cytoplasmic phosphorylation of the protein on tyrosine. The 11<em>3</em>-kilodalton ISGF-<em>3</em> protein that is phosphorylated in response to IFN-<em>alpha</em> was not phosphorylated nor translocated to the nucleus in response to IFN-gamma. Thus the two different ligands result in tyrosine phosphorylation of different combinations of latent cytoplasmic transcription factors that then act at different DNA binding sites.

Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by <em>interferon</em> gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; <em>3</em> copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-<em>alpha</em>-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.

Ubiquitously expressed <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) is directly activated after virus infection and functions as a key activator of the immediate-early <em>alpha</em>/beta <em>interferon</em> (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-<em>3</em> (IRF-<em>3</em> 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-<em>3</em>. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-<em>3</em> 5D. Among the genes upregulated by IRF-<em>3</em> were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-<em>3</em> directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-beta enhanceosome, IRF-<em>3</em> is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

BACKGROUND

Crohn's disease is associated with excess cytokine activity mediated by type 1 helper T (Th1) cells. Interleukin-12 is a key cytokine that initiates Th1-mediated inflammatory responses.

METHODS

This double-blind trial evaluated the safety and efficacy of a human monoclonal antibody against interleukin-12 (anti-interleukin-12) in 79 patients with active Crohn's disease. Patients were randomly assigned to receive seven weekly subcutaneous injections of 1 mg or <em>3</em> mg of anti-interleukin-12 per kilogram of body weight or placebo, with either a four-week interval between the first and second injection (Cohort 1) or no interruption between the two injections (Cohort 2). Safety was the primary end point, and the rates of clinical response (defined by a reduction in the score for the Crohn's Disease Activity Index [CDAI] of at least 100 points) and remission (defined by a CDAI score of 150 or less) were secondary end points.

RESULTS

Seven weeks of uninterrupted treatment with <em>3</em> mg of anti-interleukin-12 per kilogram resulted in higher response rates than did placebo administration (75 percent vs. 25 percent, P=0.0<em>3</em>). At 18 weeks of follow-up, the difference in response rates was no longer significant (69 percent vs. 25 percent, P=0.08). Differences in remission rates between the group given <em>3</em> mg of anti-interleukin-12 per kilogram and the placebo group in Cohort 2 were not significant at either the end of treatment or the end of follow-up (<em>3</em>8 percent and 0 percent, respectively, at both times; P=0.07). There were no significant differences in response rates among the groups in Cohort 1. The rates of adverse events among patients receiving anti-interleukin-12 were similar to those among patients given placebo, except for a higher rate of local reactions at injection sites in the former group. Decreases in the secretion of interleukin-12, interferon-gamma, and tumor necrosis factor alpha by mononuclear cells of the colonic lamina propria accompanied clinical improvement in patients receiving anti-interleukin-12.

CONCLUSIONS

Treatment with a monoclonal antibody against interleukin-12 may induce clinical responses and remissions in patients with active Crohn's disease. This treatment is associated with decreases in Th1-mediated inflammatory cytokines at the site of disease.

Type III <em>interferons</em> (IFNs) (interleukin-28/29 or lambda <em>interferon</em> [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/<em>3</em> in similar patterns. The IFN-lambdas were-unlike <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta)-induced directly by stimulation with IFN-<em>alpha</em> or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-<em>alpha</em> potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-<em>alpha</em> reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-<em>alpha</em>, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.

The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) system. This virus-host interplay is dependent on the production of IFN-<em>alpha</em>/beta and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor <em>3</em> (IRF<em>3</em>) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/<em>3</em>4 NS1 has a strong capacity to inhibit IRF<em>3</em> and activation of the IFN-beta promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/<em>3</em>6/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/<em>3</em>4 NS1. We identified the Phe-10<em>3</em> and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity.

Peroxisome proliferator-activated receptors (PPARs) have been implicated in metabolic diseases, such as obesity, diabetes, and atherosclerosis, due to their activity in liver and adipose tissue on genes involved in lipid and glucose homeostasis. Here, we show that the PPAR<em>alpha</em> and PPARgamma forms are expressed in differentiated human monocyte-derived macrophages, which participate in inflammation control and atherosclerotic plaque formation. Whereas PPAR<em>alpha</em> is already present in undifferentiated monocytes, PPARgamma expression is induced upon differentiation into macrophages. Immunocytochemistry analysis demonstrates that PPAR<em>alpha</em> resides constitutively in the cytoplasm, whereas PPARgamma is predominantly nuclear localized. Transient transfection experiments indicate that PPAR<em>alpha</em> and PPARgamma are transcriptionally active after ligand stimulation. Ligand activation of PPARgamma, but not of PPAR<em>alpha</em>, results in apoptosis induction of unactivated differentiated macrophages as measured by the TUNEL assay and the appearance of the active proteolytic subunits of the cell death protease caspase-<em>3</em>. However, both PPAR<em>alpha</em> and PPARgamma ligands induce apoptosis of macrophages activated with tumor necrosis factor <em>alpha</em>/<em>interferon</em> gamma. Finally, PPARgamma inhibits the transcriptional activity of the NFkappaB p65/RelA subunit, suggesting that PPAR activators induce macrophage apoptosis by negatively interfering with the anti-apoptotic NFkappaB signaling pathway. These data demonstrate a novel function of PPAR in human macrophages with likely consequences in inflammation and atherosclerosis.

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-<em>alpha</em>/beta receptor-deficient (IFN- <em>alpha</em>/betaR-/-) mice and primary neuronal cultures. IFN-<em>alpha</em>/betaR-/- mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 +/- 0.7 and <em>3</em>.8+/- 0.5 days after infection with 10(0) and 10(2) PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10(2) PFU showed 62% mortality and a MTD of 11.9 +/- 1.9 days. IFN-<em>alpha</em>/betaR-/- mice developed high viral loads by day <em>3</em> after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-<em>alpha</em>/betaR-/- mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-beta either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-<em>alpha</em>/beta controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.

To replicate in vivo, viruses must circumvent cellular antiviral defense mechanisms, including those induced by the <em>interferons</em> (IFNs). Here we demonstrate that simian virus 5 (SV5) blocks IFN signalling in human cells by inhibiting the formation of the IFN-stimulated gene factor <em>3</em> and gamma-activated factor transcription complexes that are involved in activating IFN-<em>alpha</em>/beta- and IFN-gamma-responsive genes, respectively. SV5 inhibits the formation of these complexes by specifically targeting STAT1, a component common to both transcription complexes, for proteasome-mediated degradation. Expression of the SV5 structural protein V, in the absence of other virus proteins, also inhibited IFN signalling and induced the degradation of STAT1. Following infection with SV5, STAT1 was degraded in the absence of virus protein synthesis and remained undetectable for up to 4 days postinfection. Furthermore, STAT1 was also degraded in IFN-pretreated cells, even though the cells were in an antiviral state. Since pretreatment of cells with IFN delayed but did not prevent virus replication and protein synthesis, these observations suggest that following infection of IFN-pretreated cells, SV5 remains viable within the cells until they eventually go out of the antiviral state.

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, interleukin-11, and ciliary neurotrophic factor bind to receptor complexes that share the signal transducer gp1<em>3</em>0. Upon binding, the ligands rapidly activate DNA binding of acute-phase response factor (APRF), a protein antigenically related to the p91 subunit of the <em>interferon</em>-stimulated gene factor-<em>3</em> <em>alpha</em> (ISGF-<em>3</em> <em>alpha</em>). These cytokines caused tyrosine phosphorylation of APRF and ISGF-<em>3</em> <em>alpha</em> p91. Protein kinases of the Jak family were also rapidly tyrosine phosphorylated, and both APRF and Jak1 associated with gp1<em>3</em>0. These data indicate that Jak family protein kinases may participate in IL-6 signaling and that APRF may be activated in a complex with gp1<em>3</em>0.

The Ebola virus VP<em>3</em>5 protein was previously found to act as an <em>interferon</em> (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP<em>3</em>5 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-beta) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP<em>3</em>5 inhibits virus infection-induced transcriptional activation of IFN regulatory factor <em>3</em> (IRF-<em>3</em>)-responsive mammalian promoters and that VP<em>3</em>5 does not block signaling from the IFN-<em>alpha</em>/beta receptor. The ability of VP<em>3</em>5 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-<em>3</em>, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP<em>3</em>5 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-<em>3</em>, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP<em>3</em>5 prevents the IRF-<em>3</em>-dependent activation of the IFN-<em>alpha</em>4 promoter in response to viral infection. The inhibition of IRF-<em>3</em> appears to occur through an inhibition of IRF-<em>3</em> phosphorylation. VP<em>3</em>5 blocks virus-induced IRF-<em>3</em> phosphorylation and subsequent IRF-<em>3</em> dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-<em>3</em>. These data suggest that in Ebola virus-infected cells, VP<em>3</em>5 inhibits the induction of antiviral genes, including the IFN-beta gene, by blocking IRF-<em>3</em> activation.

The <em>interferon</em> regulatory factor (IRF) family of transcription factors regulate the <em>interferon</em> (IFN) system, among which IRF-<em>3</em> is involved in the virus-induced IFN-beta gene expression. Here we show that another member IRF-7 is critical for the IFN-<em>alpha</em> gene induction. Unlike the IRF-<em>3</em> gene, the IRF-7 gene is induced by IFNs through activation of the ISGF<em>3</em> transcription factor, and IRF-7 undergoes virus-induced nuclear translocation. In cells lacking p48, an essential component of IFN stimulated gene factor <em>3</em> (ISGF<em>3</em>), ectopic expression of IRF-7 but not IRF-<em>3</em> can rescue the deficiency to induce IFN-<em>alpha</em> genes. These results indicate that IRF-7 is a key factor in the positive feedback regulation of IFN-<em>alpha</em>/beta production.