We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-␥) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-␥ inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-␥. While phosphorylation of other proteins in the IFN-␥ pathway was not affected, activation of Stat1 by other ligandreceptor systems was also blocked by VV. This block of Stat1 activation was dose dependent, and although viral protein synthesis was not required, entry and uncoating of viral cores appear to be needed to block the accumulation of phosphorylated Stat1. These results suggest that a virion component is responsible for the effect. VV virions contain a phosphatase (VH1) that is sensitive to the phosphatase inhibitor Na 3 VO 4 but not to okadaic acid. Addition of Na 3 VO 4 but not okadaic acid restored normal Stat1 phosphorylation levels in VV-infected cells. Moreover, virions containing reduced levels of VH1 were unable to block the IFN-␥ signaling pathway. In vitro studies show that the phosphatase can bind and dephosphorylate Stat1, indicating that this transcription factor can be a substrate for VH1. Our results reveal a novel mechanism by which VV interferes with the onset of host immune responses by blocking the IFN-␥ signal cascade through the dephosphorylating activity of the viral phosphatase VH1.Gamma interferon (IFN-␥) plays a key role in host defense (67). It regulates the adaptive immune response by enhancing major histocompatibility complex (MHC) class I expression in most cells and inducing MHC class II expression in antigenpresenting cells and endothelial cells. It is also the major physiological activating factor of macrophages and is responsible for induction of nonspecific immune responses. IFN-␥ acts synergistically with tumor necrosis factor (cytotoxic activity/ inflammatory response) and IFN␣/␤ (antiviral activity). IFNs are essential and functionally nonredundant in successful host responses to certain viruses (48).IFN-␥ exerts its action through its ability to bind to the IFN-␥ receptor (IFN-␥R) and induce dimerization of receptor ␣ and ␤ subunit pairs to form a heterotetramer (18,19). The IFN-␥R ␣ and ␤ chains associate with Janus protein kinases Jak1 and Jak2, respectively (34,37,47,65). Ligand-induced association of the receptor subunits allows these kinases to phosphorylate the IFN-␥R␣. The phosphorylated tyrosine (PTyr) and adjacent residues constitute a docking site for the sh2 domain of Stat1 (p91) (16,22), which is present in the cytoplasm as a latent transcription factor. Once bound to the receptor-Jak complex, Stat1 is phosphorylated (p91-P) (57) and subsequently dissociates from the receptor and homodimerizes, by a process that is not fully understood, to form the IFN-␥ activation factor GAF. The IFN-␥ activation factor is translocated to the nucleus, where it is able to bind to the IFN-␥ activation sequence-GAS-in the promoters of genes whose expression is induced by IFN-␥ (5...

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. Here we report the discovery and initial development of RSV604, a novel benzodiazepine with submicromolar anti-RSV activity. It proved to be equipotent against all clinical isolates tested of both the A and B subtypes of the virus. The compound has a low rate of in vitro resistance development. Sequencing revealed that the resistant virus had mutations within the nucleocapsid protein. This is a novel mechanism of action for anti-RSV compounds. In a three-dimensional human airway epithelial cell model, RSV604 was able to pass from the basolateral side of the epithelium effectively to inhibit virus replication after mucosal inoculation. RSV604, which is currently in phase II clinical trials, represents the first in a new class of RSV inhibitors and may have significant potential for the effective treatment of RSV disease.Human respiratory syncytial virus (RSV) is the most important respiratory pathogen that causes lower respiratory tract infections, such as bronchiolitis and pneumonia, in infants and young children, resulting in up to 125,000 hospitalizations annually in the United States (3). The infants most at risk of severe disease are those under 6 weeks of age, those with bronchopulmonary dysplasia, congenital heart disease, or immunodeficiency, and those born prematurely. Hospital admission rates for these groups range between 5% and 30% (20,25). The mortality rate among children admitted to hospital is approximately 3% for those with heart and lung problems and up to 1% for those without these risk factors (11,25). In adults and the elderly, RSV pneumonia is increasingly recognized as a significant cause of morbidity and mortality, being associated with more than 17,000 deaths annually between 1991 and 1998 (9, 22). Among the hospitalized elderly, mortality can be as high as 10 to 20%, and among severely immunocompromised patients with RSV pneumonia, it can be on the order of 50 to 70% (10). There is therefore an urgent and unmet medical need for novel therapies to deal with infections caused by this virus.The development of new therapeutics for RSV was recently reviewed (17,19). Although research into the prevention and treatment of RSV infection has been ongoing for almost 40 years, vaccine development is difficult (8, 13), and to date, there is no clinically approved vaccine. The development of RSV vaccines for use in young infants has been complicated by reduced immune responses in this age group due to immunologic immaturity and the immunosuppressive effects of maternal antibodies. Passive immunization with the monoclonal antibody palivizumab (Synagis) has provided about 50% protection to high-risk children (21). These include infants born prematurely and those with congenital conditions. Because the antibody has to be given prophylactically and treatment is very expensive, its use is limited mainly to developed countries. The effect...

We showed previously that type I interferon causes a down-regulation of mitochondrial gene expression. We show here that IFN treatment leads to functional impairment of mitochondria. Western blot analysis indicated that interferon treatment reduces the steady-state level of cytochrome b in murine L-929 cells. Interferon produced a reduction in cytochrome c oxidase and NADH-cytochrome c reductase activities of isolated mitochondria as well as inhibiting electron transport in isolated mitochondria and in intact cells. Several mitochondrial mRNAs are affected by interferon treatment in human Daudi lymphoblastoid cells, which are highly sensitive to the antiproliferative effects of interferon. Electron transport in Daudi cells was also inhibited by interferon both in intact cells and isolated mitochondria with a dose response identical to that for the antiproliferative response. In contrast, a Daudi strain resistant to the antiproliferative effects of interferon showed no down-regulation of mRNA expression and no inhibition of electron transport. Possibly as a consequence of the inhibitory effect on mitochondrial gene expression, treatment with interferon causes a reduction in cellular ATP levels. The inhibition of cellular growth by interferon may thus be partly a consequence of a reduction in cellular ATP levels.

The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts ␥-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.Nucleoside diphosphate kinases (NDPKs) play a key role in the maintenance of intracellular pools of deoxynucleoside triphosphates (dNTPs) and NTPs via the transfer of phosphate from an NTP donor to an NDP acceptor. In addition, certain variants of these enzymes are involved in a variety of cellular processes unrelated to their catalytic activity, such as differentiation, proliferation, and suppression of tumor metastasis (8).In particular, nm23-H2/NDPK B has been identified as a DNA-binding protein and transcriptional activator of the human c-myc gene, previously known as PuF (29,31). NDPKs are typically intracellular enzymes, although recently an ectoenzyme has been detected on the surface of mammalian cells (19,20), and NDPKs have been reported to be secreted by the prokaryotic pathogens Mycobacterium bovis, Pseudomonas aeruginosa, and Vibrio cholerae (32,38,39).Trichinella spiralis is a ubiquitous nematode parasite of a wide variety of mammalian species, including humans, and is remarkable among multicellular parasites in adopting an intracellular habitat, both in the systemic phase of infection in skeletal muscle cells and in the enteral phase, in which it invades and migrates through mucosal epithelial cells (9). It is likely that secreted products are involved in survival and development of parasites in both environments. Consistent with this assumption, infective larvae possess a large organelle termed the stichosome, which is the major source of secreted proteins which can be recovered from in vitro culture of parasites (9).We have previously demonstrated that T. spiralis infective larvae secrete serine/threonine protein kinases (3). During the course of these studies, it became apparent that phosphorylation of a protein with an estimated mass of 17 kDa was regulated independently of both exogenous substrates and the major endogenous parasite substrate for protein kinase activity, a doublet of 50 and 55 kDa. We hypothesized that this may result from the activity of an additional enzyme and demonstrate here that T. spiralis se...

Although Vaccinia virus (VACV) was used to eradicate smallpox by dermal vaccination, there is little information available about the immune response induced at the vaccination site. Previously, an intradermal murine model that mimics smallpox vaccination was established. Here, this model was used to investigate which leukocytes are recruited to the infected lesion and what are the kinetics of recruitment. Data presented show that VACV infection induced the infiltration of macrophages, followed by granulocytes and lymphocytes. Up to 4 days post-infection, the major lymphocyte population was TCRcd T cells, but thereafter, there was a large recruitment of CD4 + and CD8 + T cells. Interestingly, the majority of T cells expressed the natural killer-cell marker DX5. This report is the first to characterize the local immune response sequence to VACV infection and represents a benchmark against which the responses induced by genetically modified VACVs may be compared.Vaccinia virus (VACV) is a potent vaccine against smallpox, but its use was discontinued in the 1970s following smallpox eradication. Recently, vaccination of limited numbers of health-care workers was introduced in the USA, the UK and elsewhere in response to a perceived threat of bioterrorism with Variola virus, the aetiological agent of smallpox. Despite worldwide use of VACV as the smallpox vaccine, only recently have studies analysed the systemic immune response induced in animals (Belyakov et al., 2003;Earl et al., 2004;Xu et al., 2004) and humans (Crotty et al., 2003;Frey et al., 2003;Hammarlund et al., 2003;Amara et al., 2004;Belshe et al., 2004; Combadiere et al., 2004;Greenberg et al., 2005; Pütz et al., 2005; Rock et al., 2005) by modern immunological techniques. However, the immune response at the vaccination site remains poorly understood.Previously, an intradermal model in the mouse ear pinnae that mimics dermal vaccination with VACV was developed (Tscharke & Smith, 1999). Following vaccination, no signs of systemic illness were observed and there was little or no virus spread from the ear. Subsequently, this model was used to assess virulence of different strains of VACV, other orthopoxviruses and VACV mutants engineered to lack specific virus genes . Fig. 1(a) shows that this vaccination induced protection against challenge with all doses of VACV strain Western Reserve (WR) tested (up to 10 7 p.f.u.). It was established previously that doses of 10 5 p.f.u. or greater are lethal in this model (Wilcock et al., 1999;Symons et al., 2002;Fogg et al., 2004;Davies et al., 2005). Another report analysed cells that had infiltrated into VACV-infected ears by separation of the ventral and the dorsal dermal sheets and incubation of these on culture medium at 37 uC for 8-10 h (Reading & Smith, 2003). Non-adherent cells that had migrated from the dermal layers were pooled with the loosely adherent cells recovered by a further incubation (20 min) in PBS containing glucose (2 mg ml 21 ), but without calcium and magnesium. With this technique, called the '...