scholarly journals Vaccinia Virus Blocks Gamma Interferon Signal Transduction: Viral VH1 Phosphatase Reverses Stat1 Activation

2001 ◽  
Vol 75 (7) ◽  
pp. 3185-3196 ◽  
Author(s):  
Pilar Najarro ◽  
Paula Traktman ◽  
John A. Lewis
Keyword(s):  
Signal Transduction ◽  
Vaccinia Virus ◽  
Okadaic Acid ◽  
Nuclear Translocation ◽  
Gamma Interferon ◽  
Viral Protein Synthesis ◽  
Host Immune Responses ◽  
Infected Cells ◽  
Ifn Γ

ABSTRACT We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-γ) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-γ inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-γ. While phosphorylation of other proteins in the IFN-γ pathway was not affected, activation of Stat1 by other ligand-receptor 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 Na3VO4 but not to okadaic acid. Addition of Na3VO4 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-γ 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-γ signal cascade through the dephosphorylating activity of the viral phosphatase VH1.

scholarly journals The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model

2002 ◽  
Vol 83 (11) ◽  
pp. 2833-2844 ◽  
Author(s):  
Julian A. Symons ◽  
Elizabeth Adams ◽  
David C. Tscharke ◽  
Patrick C. Reading ◽  
Herman Waldmann ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Deletion Mutant ◽  
Soluble Factor ◽  
Gene Encoding ◽  
Mouse Splenocytes ◽  
Infected Cells ◽  
Ifn Γ ◽  
Revertant Virus

A bioassay that measured the interleukin (IL)-12-induced production of interferon (IFN)-γ from mouse splenocytes was used to identify a soluble factor in the supernatants of vaccinia virus (VV)-infected cells that inhibited the production of IFN-γ. This soluble factor was expressed by 14 out of 16 VV strains including the Western Reserve (WR) strain, but strains Copenhagen and Tashkent and a mutant of strain WR called 6/2 lacked this activity. The gene encoding this activity was identified as C12L by transferring DNA present in VV WR but missing in VV WR 6/2 into VV Copenhagen and testing for expression of the soluble factor. The C12L protein shows amino acid similarity to IL-18 binding proteins that are encoded by poxviruses, mice and humans, and C12L protein produced from VV or baculovirus inhibited the biological activity of mouse IL-18 in vitro. Thus the inhibition of IL-12-induced IFN-γ production was due to indirect effects of C12L on IL-18, illustrating the synergistic action of these pro-inflammatory cytokines. To study the role of the C12L protein in the virus life-cycle, we constructed a deletion mutant lacking the C12L gene and a revertant virus in which the gene was reinserted into the deletion mutant. In vitro the replication and plaque size of these viruses were indistinguishable. However, infection of BALB/c mice by the intranasal route showed that the deletion mutant was attenuated and induced lower weight loss and signs of illness compared to controls.

scholarly journals Vaccinia Virus Vectors with an Inactivated Gamma Interferon Receptor Homolog Gene (B8R) Are Attenuated In Vivo without a Concomitant Reduction in Immunogenicity

2001 ◽  
Vol 75 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Paulo H. Verardi ◽  
Leslie A. Jones ◽  
Fatema H. Aziz ◽  
Shabbir Ahmad ◽  
Tilahun D. Yilma
Keyword(s):  
Antiviral Activity ◽  
Vaccinia Virus ◽  
Gamma Interferon ◽  
Surprising Result ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Concomitant Reduction ◽  
Ifn Γ

ABSTRACT The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-γ) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-γ, including human and rat IFN-γ; it does not, however, bind significantly to murine IFN-γ. Here we report on the construction and characterization of recombinant VVs (rVVs) lacking the B8R gene. While the deletion of this gene had no effect on virus replication in vitro, rVVs lacking the B8R gene were attenuated for mice. There was a significant decrease in weight loss and mortality in normal mice, and nude mice survived significantly longer than did controls inoculated with parental virus. This is a surprising result considering the minimal binding of the B8R protein to murine IFN-γ and its failure to block the antiviral activity of this cytokine in vitro. Such reduction in virulence could not be determined in rats, since they are considerably more resistant to VV infection than are mice. Finally, deletion of the B8R gene had no detectable effects on humoral immune responses. Mice and rats vaccinated with the rVVs showed identical humoral responses to both homologous and heterologous genes expressed by VV. This study demonstrates that the deletion of the VV B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.

scholarly journals Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein

2005 ◽  
Vol 79 (9) ◽  
pp. 5632-5639 ◽  
Author(s):  
Chulbul M. I. Ahmed ◽  
Marjorie A. Burkhart ◽  
Prem S. Subramaniam ◽  
Mustafa G. Mujtaba ◽  
Howard M. Johnson
Keyword(s):  
Antiviral Activity ◽  
Vaccinia Virus ◽  
Virulence Factor ◽  
Nuclear Translocation ◽  
Extracellular Domain ◽  
Gamma Interferon ◽  
Peptide Mimetics ◽  
Direct Role ◽  
African Green Monkey Kidney ◽  
Ifn Γ

ABSTRACT We have developed peptide mimetics of gamma interferon (IFN-γ) that play a direct role in the activation and nuclear translocation of STAT1α transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-γ receptor but rather bind to the cytoplasmic domain of the receptor chain 1, IFNGR-1, and thereby initiate the cellular signaling. Thus, we hypothesized that these mimetics would bypass the poxvirus virulence factor B8R protein that binds to intact IFN-γ and prevents its interaction with the receptor. Human and murine IFN-γ mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN-γ mimetic peptide was also expressed via chemical synthesis with an attached lipophilic group for penetration of cell plasma membrane. In contrast to intact human IFN-γ, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN-γ receptor extracellular domain. Expression of B8R protein in WISH cells did not block the antiviral effect of the mimetics against encephalomyocarditis or vesicular stomatitis virus, while the antiviral activity of human IFN-γ was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN-γ mimetics was not affected by B8R protein, while IFN-γ-induced upregulation was blocked. Finally, the mimetics, but not IFN-γ, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN-γ can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.

scholarly journals A Cytomegalovirus Inhibitor of Gamma Interferon Signaling Controls Immunoproteasome Induction

2004 ◽  
Vol 78 (4) ◽  
pp. 1831-1842 ◽  
Author(s):  
Selina Khan ◽  
Albert Zimmermann ◽  
Michael Basler ◽  
Marcus Groettrup ◽  
Hartmut Hengel
Keyword(s):  
Mhc Class I ◽  
Gamma Interferon ◽  
Class I ◽  
Peptide Transport ◽  
Antigenic Peptides ◽  
Cell Repertoire ◽  
Mcmv Infection ◽  
Infected Cells ◽  
Ifn Γ

ABSTRACT Both human and mouse cytomegaloviruses (HCMV and MCMV) avoid peptide presentation through the major histocompatibility complex (MHC) class I pathway to CD8+ T cells. Within the MHC class I pathway, the vast majority of antigenic peptides are generated by the proteasome system, a multicatalytic protease complex consisting of constitutive subunits, three of which can be replaced by enzymatically active gamma interferon (IFN-γ)-inducible subunits, i.e., LMP2, LMP7, and MECL1, to form the so-called immunoproteasomes. Here, we show that steady-state levels of immunoproteasomes are readily formed in response to MCMV infection in the liver. In contrast, the incorporation of immunoproteasome subunits was prevented in MCMV-infected, as well as HCMV-infected, fibroblasts in vitro. Likewise, the expression of the IFN-γ-inducible proteasome regulator PA28αβ was also impaired in MCMV-infected cells. Both MCMV and HCMV did not alter the constitutive-subunit composition of proteasomes in infected cells. Quantitative assessment of LMP2, MECL1, and LMP7 transcripts revealed that the inhibition of immunoproteasome formation occurred at a pretranscriptional level. Remarkably, a targeted deletion of the MCMV gene M27, encoding an inhibitor of STAT2 that disrupts IFN-γ receptor signaling, largely restored transcription and protein expression of immunoproteasome subunits in infected cells. While CMV block peptide transport and MHC class I assembly by posttranslational strategies, immunoproteasome assembly, and thus the repertoire of proteasomal peptides, is controlled by pretranscriptional mechanisms. We hypothesize that the blockade of immunoproteasome formation has considerable consequences for shaping the CD8+-T-cell repertoire during the effector phase of the immune response.

scholarly journals Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes

2007 ◽  
Vol 75 (11) ◽  
pp. 5338-5345 ◽  
Author(s):  
Kee-Jong Hong ◽  
Jason R. Wickstrum ◽  
Hung-Wen Yeh ◽  
Michael J. Parmely
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Gamma Interferon ◽  
Interferon Response ◽  
Interleukin 12 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

scholarly journals The Linker Domain of Stat1 Is Required for Gamma Interferon-Driven Transcription

1999 ◽  
Vol 19 (7) ◽  
pp. 5106-5112 ◽  
Author(s):  
Edward Yang ◽  
Zilong Wen ◽  
Richard L. Haspel ◽  
Jue J. Zhang ◽  
James E. Darnell
Keyword(s):  
Dna Binding ◽  
Nuclear Translocation ◽  
Contact Point ◽  
Sh2 Domain ◽  
Gamma Interferon ◽  
Transactivation Domain ◽  
Dimerization Domain ◽  
Transcriptional Responses ◽  
Ifn Γ ◽  
Linker Domain

ABSTRACT Upon binding of gamma interferon (IFN-γ) to its receptor, the latent transcription factor Stat1 becomes phosphorylated, dimerizes, and enters the nucleus to activate transcription. In response to IFN-α, Stat1 binds to Stat2 in a heterodimer that recruits p48, an IRF family member, to activate transcription. A number of functional domains of the STATs, including a C-terminal transactivation domain, a dimerization domain, and an SH2 domain, are known. However, the highly conserved residues between the DNA binding and SH2 domains (463 to 566), recently christened the linker domain on the basis of crystallographic studies, have remained without a known function. In the present study, we report that KE544-545AA point mutants in Stat1 abolish transcriptional responses to IFN-γ but not to IFN-α. We further show that this mutant Stat1 undergoes normal phosphorylation, nuclear translocation, and DNA binding. Taken together with recent structural evidence, these results suggest that the linker domain acts as a critical contact point during the construction of a Stat1-driven transcriptional complex.

scholarly journals Gamma Interferon Mediates Experimental Cerebral Malaria by Signaling within Both the Hematopoietic and Nonhematopoietic Compartments

2017 ◽  
Vol 85 (11) ◽  
Author(s):  
Ana Villegas-Mendez ◽  
Patrick Strangward ◽  
Tovah N. Shaw ◽  
Ivana Rajkovic ◽  
Vinko Tosevski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cerebral Malaria ◽  
Myeloid Cell ◽  
Gamma Interferon ◽  
Cell Populations ◽  
Experimental Cerebral Malaria ◽  
Cerebral Pathology ◽  
Multiple Cell ◽  
Ifn Γ

ABSTRACT Experimental cerebral malaria (ECM) is a gamma interferon (IFN-γ)-dependent syndrome. However, whether IFN-γ promotes ECM through direct and synergistic targeting of multiple cell populations or by acting primarily on a specific responsive cell type is currently unknown. Here, using a panel of cell- and compartment-specific IFN-γ receptor 2 (IFN-γR2)-deficient mice, we show that IFN-γ causes ECM by signaling within both the hematopoietic and nonhematopoietic compartments. Mechanistically, hematopoietic and nonhematopoietic compartment-specific IFN-γR signaling exerts additive effects in orchestrating intracerebral inflammation, leading to the development of ECM. Surprisingly, mice with specific deletion of IFN-γR2 expression on myeloid cells, T cells, or neurons were completely susceptible to terminal ECM. Utilizing a reductionist in vitro system, we show that synergistic IFN-γ and tumor necrosis factor (TNF) stimulation promotes strong activation of brain blood vessel endothelial cells. Combined, our data show that within the hematopoietic compartment, IFN-γ causes ECM by acting redundantly or by targeting non-T cell or non-myeloid cell populations. Within the nonhematopoietic compartment, brain endothelial cells, but not neurons, may be the major target of IFN-γ leading to ECM development. Collectively, our data provide information on how IFN-γ mediates the development of cerebral pathology during malaria infection.

scholarly journals Gamma Interferon Production, but Not Perforin-Mediated Cytolytic Activity, of T Cells Is Required for Prevention of Toxoplasmic Encephalitis in BALB/c Mice Genetically Resistant to the Disease

2004 ◽  
Vol 72 (8) ◽  
pp. 4432-4438 ◽  
Author(s):  
Xisheng Wang ◽  
Hoil Kang ◽  
Takane Kikuchi ◽  
Yasuhiro Suzuki
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nude Mice ◽  
Genetic Resistance ◽  
Cytolytic Activity ◽  
Gamma Interferon ◽  
Toxoplasmic Encephalitis ◽  
Wild Type ◽  
Ifn Γ

ABSTRACT We previously showed the requirement of both T cells and gamma interferon (IFN-γ)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-γ production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-γ knockout (IFN-γ−/−), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-γ-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-γ−/− mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-γ after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-γ mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-γ production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-γ production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.

scholarly journals Modulation of GAPDH expression and cellular localization after vaccinia virus infection of human adherent monocytes.

2003 ◽  
Vol 50 (3) ◽  
pp. 667-676 ◽  
Author(s):  
Krystyna W Nahlik ◽  
Anna K Mleczko ◽  
Magdalena K Gawlik ◽  
Hanna B Rokita
Keyword(s):  
Vaccinia Virus ◽  
Cellular Localization ◽  
Nuclear Translocation ◽  
Cell Types ◽  
Glycolytic Enzyme ◽  
Gene Transcript ◽  
Gapdh Expression ◽  
Gapdh Mrna ◽  
Infected Cells ◽  
Novel Target

Vaccinia virus is able to replicate in many cell types and is known to modulate apoptosis in infected cells. In this study, expression of apoptosis-related genes was screened in human adherent monocytes after vaccinia infection using a DNA array. A marked increase of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was found. Increased expression and nuclear translocation of GAPDH have recently been reported to participate in apoptosis of many cell types. To confirm the array results, levels of GAPDH mRNA were estimated by RT-PCR, showing an increase at 4 h p.i. followed by a slight decrease, which correlated with the viral anti-apoptotic E3L gene transcript levels. Subcellular localization of the enzyme in human monocytes was examined by Western blot and immunostaining of the infected cells. Both experiments revealed accumulation of GAPDH in the nucleus at 14 h p.i., which was completely suppressed at 24 h p.i. This might indicate GAPDH as a novel target for vaccinia anti-apoptotic modulation.

scholarly journals Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection ofMycobacterium tuberculosis Infection

1998 ◽  
Vol 5 (4) ◽  
pp. 531-536 ◽  
Author(s):  
Nuket Desem ◽  
Stephen L. Jones
Keyword(s):  
Enzyme Immunoassay ◽  
Skin Test ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Tuberculin Skin Testing ◽  
In Vitro Test ◽  
Detection Range ◽  
Ifn Γ

ABSTRACT A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.

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