2fTGH Cells Research Articles

Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

UMP-CMP kinase 2 inhibits ZIKV replication through activation of type I IFN signaling pathway.

Cytidine/uridine monophosphate kinase 2 (UMP-CMP kinase 2, CMPK2) has been reported as an antiviral interferon-stimulated gene (ISG). We previously observed that the expression of CMPK2 was significantly upregulated after Zika Virus (ZIKV) infection in A549 cells. However, the association and the underlying mechanisms between CMPK2 induction and ZIKV replication remain to be determined. We investigated the induction of CMPK2 during ZIKV infection and the effect of CMPK2 on ZIKV replication in A549, U251, Vero, IFNAR-deficient U5A and its parental 2fTGH cells, Huh7 and its RIG-I-deficient derivatives Huh7.5.1 cells. The activation status of Jak-STAT signaling pathway was determined by detecting the phosphorylation level of STAT1, the activity of interferon stimulated response element (ISRE) and the expression of several interferon stimulated genes (ISGs). We found that ZIKV infection induced CMPK2 expression through an IFNAR and RIG-I dependent manner. Overexpression of CMPK2 inhibited while CMPK2 knockdown promoted ZIKV replication in A549 and U251 cells. Mechanically, we found that CMPK2 overexpression increased IFNβ expression and activated Jak/STAT signaling pathway as shown by the increased level of p-STAT1, enhanced activity of ISRE, and the upregulated expression of downstream ISGs. These findings suggest that ZIKV infection induced CMPK2 expression, which inhibited ZIKV replication and serves as a positive feedback regulator for IFN-Jak/STAT pathway.

Phosphorylation of interferon regulatory factor 9 (IRF9).

IRF9 is a transcription factor that mediates the expression of interferon-stimulated genes (ISGs) through the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway. The JAK-STAT pathway is regulated through phosphorylation reactions, in which all components of the pathway are known to be phosphorylated except IRF9. The enigma surrounding IRF9 regulation by a phosphorylation event is intriguing. As IRF9 plays a major role in establishing an antiviral state in host cells, the topic of IRF9 regulation warrants deeper investigation. Initially, total lysates of 2fTGH and U2A cells (transfected with recombinant IRF9) were filter-selected and concentrated using phosphoprotein enrichment assay. The phosphoprotein state of IRF9 was further confirmed using Phos-tag™ assay. All protein expression was determined using Western blotting. Tandem mass spectrometry was conducted on immunoprecipitated IRF9 to identify the phosphorylated amino acids. Finally, site-directed mutagenesis was performed and the effects of mutated IRF9 on relevant ISGs (i.e., USP18 and Mx1) was evaluated using qPCR. IRF9 is phosphorylated at S252 and S253 under IFNβ-induced condition and R242 under non-induced condition. Site-directed mutagenesis of S252 and S253 to either alanine or aspartic acid has a modest effect on the upregulation of USP18 gene-a negative regulator of type I interferon (IFN) response-but not Mx1 gene. Our preliminary study shows that IRF9 is phosphorylated and possibly regulates USP18 gene expression. However, further in vivo studies are needed to determine the significance of IRF9 phosphorylation.

IRF2 inhibits ZIKV replication by promoting FAM111A expression to enhance the host restriction effect of RFC3

BackgroundAlthough interferon regulatory factor 2 (IRF2) was reported to stimulate virus replication by suppressing the type I interferon signaling pathway, because cell cycle arrest was found to promote viral replication, IRF2-regulated replication fork factor (FAM111A and RFC3) might be able to affect ZIKV replication. In this study, we aimed to investigate the function of IRF2, FAM111A and RFC3 to ZIKV replication and underlying mechanism.MethodssiIRF2, siFAM111A, siRFC3 and pIRF2 in ZIKV-infected A549, 2FTGH and U5A cells were used to explore the mechanism of IRF2 to inhibit ZIKV replication. In addition, their expression was analyzed by RT-qPCR and western blots, respectively.ResultsIn this study, we found IRF2 expression was increased in ZIKV-infected A549 cells and IRF2 inhibited ZIKV replication independent of type I IFN signaling pathway. IRF2 could activate FAM111A expression and then enhanced the host restriction effect of RFC3 to inhibit replication of ZIKV.ConclusionsWe speculated the type I interferon signaling pathway might not play a leading role in regulating ZIKV replication in IRF2-silenced cells. We found IRF2 was able to upregulate FAM111A expression and thus enhance the host restriction effect of RFC3 on ZIKV.

STAT2-dependent induction of RNA adenosine deaminase ADAR1 by type I interferon differs between mouse and human cells in the requirement for STAT1

Expression of adenosine deaminase acting on RNA1 (ADAR1) is driven by alternative promoters. Promoter PA, activated by interferon (IFN), produces transcripts that encode the inducible p150 ADAR1 protein, whereas PB specifies the constitutively expressed p110 protein. We show using Stat1−/−, Stat2−/− and IRF9−/− MEFs that induction of ADAR1 p150 occurs by STAT2- and IRF9-dependent signaling that is enhanced by, but not obligatorily dependent upon, STAT1. Chromatin immunoprecipitation analysis demonstrated STAT2 at the PA promoter in IFN-treated Stat1−/− cells, whereas IFN-treated wild-type cells showed both STAT1 and STAT2 bound at PA. By contrast, with human 2fTGH cells and mutants U3A or U6A, ADAR1 induction by IFN was dependent upon both STAT1 and STAT2. These results suggest that transcriptional activation of Adar1 by IFN occurs in the absence of STAT1 by a non-canonical STAT2-dependent pathway in mouse but not human cells.

A role for vaccinia virus protein C16 in reprogramming cellular energy metabolism.

Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50 % may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1α by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1α is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vΔC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vΔC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1α.

Characterization of distinct sub-cellular location of transglutaminase type II: changes in intracellular distribution in physiological and pathological states.

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities unrelated to its originally identified functions. Apart from post-translational modifications of proteins (peculiar to the transglutaminase family enzymes), TG2 is involved in diverse biological functions, including cell death, signaling, cytoskeleton rearrangements, displaying enzymatic activities, G-protein and non-enzymatic biological functions. It is involved in a variety of human diseases such as celiac disease, diabetes, neurodegenerative diseases, inflammatory disorders and cancer. Regulatory mechanisms might exist through which cells control multifunctional protein expression as a function of their sub-cellular localization. The definition of the tissue and cellular distribution of such proteins is important for the determination of their function(s). We investigate the sub-cellular localization of TG2 by confocal and immunoelectron microscopy techniques in order to gain an understanding of its properties. The culture conditions of human sarcoma cells (2fTGH cells), human embryonic kidney cells (HEK293TG) and human neuroblastoma cells (SK-n-BE(2)) are modulated to induce various stimuli. Human tissue samples of myocardium and gut mucosa (diseased and healthy) are also analyzed. Immuno-gold labeling indicates that TG2 is localized in the nucleus, mitochondria and endoplasmic reticulum under physiological conditions but that this is not a stable association, since different locations or different amounts of TG2 can be observed depending on stress stimuli or the state of activity of the cell. We describe a possible unrecognized location of TG2. Our findings thus provide useful insights regarding the functions and regulation of this pleiotropic enzyme.

Type I IFN-independent RIG-I expression in response to double-stranded RNA (P1399)

Abstract Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor and is included in a RIG-I-like receptors (RLRs) family. Following invasion of RNA viruses into host cells, RIG-I senses the viral nucleic acids, leading to activation of antiviral innate immune responses. RIG-I itself is known as an IFN-inducible gene; however, little is known about how RIG-I is transcriptionally regulated. In this study, we studied molecular mechanisms of the expression of RIG-I in response to dsRNA. To investigate involvement of type I IFN-mediated pathway in the expression of RIG-I in response to dsRNA, we transfected with polyinosinic-polycytidylic acid (polyI:C), a synthetic dsRNA in STAT1-deficient U3A cells and type I IFN receptor-deficient U5A cells. Both type I and type II IFN are known to induce RIG-I, suggesting involvement of STAT1 in the RIG-I expression. However, the expression of RIG-I was observed in both U3A cells and their parental 2fTGH cells in response to polyI:C. We also found that the expression of RIG-I was induced in U5A cells with a slightly different kinetic. These results suggested that RIG-I expression is mainly mediated by the induction of type I IFN followed by STAT1 phosphorylation. In addition, to “fine-tuning” the RIG-I expression in response to viral infection, other molecules may participate in the induction of RIG-I.

Septic sera induces apoptosis and DNA fragmentation factor 40 activation in fibroblasts

Sepsis, the systemic response to infection, is the leading cause of death in the intensive care units worldwide. Septic patients can succumb through the development of early refractory hypotension or late multiple organ dysfunction. Misregulation of apoptosis during sepsis may contribute to cellular dysfunction and multiple organ dysfunction. Utilizing a tissue culture model which mimics the human disease, we demonstrate that the addition of sera derived from septic patients induces apoptosis in human fibroblast cells. Addition of septic sera to 2fTGH cells induced apoptosis by activating caspase 8, caspase 3 and DNA fragmentation factor 40 (DFF 40). Interestingly, the addition of septic sera to cells which lack STAT1 (U3A cells) did not activate DFF 40. U3A cells were also shown to be resistant to septic serum induced apoptosis. These data suggest that DFF 40 mediated apoptosis plays a significant role in mediating sepsis induced cellular dysfunction.

Pregnancy and interferon tau regulate N-myc interactor in the ovine uterus

In ruminants, interferon tau (IFNT) is synthesized and secreted by the mononuclear trophectoderm cells of the conceptus and maintains the corpus luteum and its secretion of progesterone for successful implantation and maintenance of pregnancy. In this study, we examined regulation of the expression of N-myc interactor ( NMI) gene by IFNT in the ovine uterus based on results of microarray data from a study that compared gene expression by human 2fTGH and U3A (STAT1-null 2fTGH) cell lines in response to treatment with IFNT or vehicle. In the present study, semiquantitative reverse transcription-polymerase chain reaction analyses verified that IFNT stimulated expression of NMI mRNA in 2fTGH (ie, in a STAT1-dependent manner), but not in U3A (STAT1-null) cells. Furthermore, results of western blot analyses indicated that immunoreactive NMI proteins in 2fTGH and U3A cell lines increased in a time-dependent manner only in response to IFNT. In ovine endometria, steady-state levels of NMI mRNA increased between days 14 and 16 of pregnancy and then decreased slightly by day 20, but there was no effect of day of the estrous cycle. Expression of NMI mRNA was most abundant in endometrial stromal cells, glandular epithelium, and conceptus trophectoderm. Intrauterine infusion of IFNT in cyclic ewes increased expression of NMI in the endometrium. Expression of NMI in ovine and bovine uterine cell lines increased in response to IFNT. Collectively, the results of the present study indicate that IFNT regulates expression of NMI mRNA and protein in ovine endometria during pregnancy via a STAT1-dependent cell signaling pathway.

Pregnancy and interferon τ regulate DDX58 and PLSCR1 in the ovine uterus during the peri-implantation period

Interferon τ (IFNT), the pregnancy recognition signal in ruminants, abrogates the luteolytic mechanism for maintenance of the corpus luteum for production of progesterone (P(4)). This study examined the expression of DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58) and phospholipid scramblase 1 (PLSCR1) mRNAs in the ovine uterus as these genes were increased most in 2fTGH (STAT1 positive) cells by IFNT. The results of this study indicated that IFNT regulates expression of DDX58 and PLSCR1 mRNAs in the ovine uterus, which confirmed the results of the in vitro transcriptional profiling experiment with the 2fTGH (parental STAT1 positive) and U3A (STAT1 null) cell lines. Steady-state levels of DDX58 and PLSCR1 mRNAs increased in cells of the ovine uterus between days 12 and 20 of pregnancy, but not between days 10 and 16 of the estrous cycle. The expression of DDX58 and PLSCR1 mRNAs was greatest in endometrial stromal cells, but there was transient expression in uterine luminal and superficial glandular epithelial cells. P(4) alone did not induce expression of DDX58 and PLSCR1 mRNAs; however, intrauterine injections of IFNT did induce expression of DDX58 and PLSCR1 mRNAs in the endometria of nonpregnant ewes independent of effects of P(4). These results indicate that IFNT induces expression of DDX58 and PLSCR1 in ovine endometrial cells via the classical STAT1-mediated cell signaling pathway. Based on their known biological effects, DDX58 and PLSCR1 are IFN-stimulated genes, which may increase the antiviral status of cells of the pregnant uterus to protect against viral infection and/or enhance secretion of type I IFNs that inhibit viral replication.

A screening method for identifying disruptions in interferon signaling reveals HCV NS3/4a disrupts Stat-1 phosphorylation

Viruses have evolved mechanisms to inhibit the innate immune response to infection. The aim of this study was to develop an efficient screening method to identify viral proteins and their ability to block Jak–Stat signaling using hepatitis C virus (HCV) as an example. The 2FTGH cell assay system was used in combination with transient transfection of HCV proteins in this study. Using 1000 U/ml IFN and 30 mM 6-TG to treat 2FTGH cells, it was established that transient protein expression in this cell system yielded 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 resulted in 22% cell survival, consistent with previous reports, while expression of the HCV serine protease NS3/4a resulted in 54% cell survival. NS3/4a was subsequently shown to inhibit phosphorylation of Stat-1 at the serine residue 727. Conclusion: the 2FTGH cell assay system can be adapted for transient screening to examine the ability of viral proteins or other potential inhibitors to block the Jak–Stat signaling pathway. We show that HCV NS3/4a is able to block this pathway at the stage of Stat-1 serine 727 phosphorylation.

Catalytic turnover of STAT1 allows PIV5 to dismantle the interferon-induced anti-viral state of cells

A dynamic model of STAT1 degradation by the V protein of parainfluenza virus 5 (PIV5; formerly SV5) has been proposed. In it, the V protein functions as a bipartite adaptor linking DDB1, a component of a cellular SCF-like ubiquitin E3 ligase complex, to STAT2, which in turn binds STAT1 and presents STAT1 to the E3 ligase complex for ubiquitination and subsequent degradation. Furthermore, it appears that loss of STAT1 from the complex results in decreased affinity of V for STAT2 such that STAT2 either dissociates from V or is displaced by STAT1/STAT2 complexes, facilitating the cycling of the DDB1/PIV5 V containing E3 complex for further rounds of STAT1 ubiquitination and degradation. By determining the approximate number of molecules of V, DDB1, STAT1 and STAT2 present in IFN-treated 2fTGH cells, we provide additional evidence for this dynamic model of STAT1 degradation. These results show that (i) in IFN-treated cells there is approximately 4-fold less STAT2 and 15-fold less DDB1 than STAT1 per cell and thus DDB1 and STAT2 must repeatedly acquire more STAT1 for degradation to go to completion, and (ii) approximately 600 molecules of V protein per cell can target as many as 120,000 molecules of STAT1 for degradation in the absence of either viral or cellular protein synthesis. The importance of this mechanism in terms of the ability of the virus to dismantle the IFN-induced anti-viral state of cells is discussed.

Importance of the Anti-Interferon Capacity of Sendai Virus C Protein for Pathogenicity in Mice

The Sendai virus (SeV) C protein blocks signal transduction of interferon (IFN), thereby counteracting the antiviral actions of IFN. Using HeLa cell lines expressing truncated or mutated SeV C proteins, we found that the C-terminal half has anti-IFN capacity, and that K(151)A, E(153)A, and R(154)A substitutions in the C protein eliminated this capacity. Here, we further created the mutant virus SeV Cm*, in which K(151)A, E(153)K, and R(157)L substitutions in the C protein were introduced without changing the amino acid sequence of overlapped P, V, and W proteins. SeV Cm* was found to lack anti-IFN capacity, as expected. While the growth rate and final yield of SeV Cm* were inferior to those of the wild-type SeV in IFN-responsive, STAT1-positive 2fTGH cells, SeV Cm* grew equivalently to the wild-type SeV in IFN-nonresponsive, STAT1-deficient U3A cells. SeV Cm* was thus shown to maintain multiplication capacity, except that it lacked anti-IFN capacity. Intranasally inoculated SeV Cm* could propagate in the lungs of STAT1(-/-) mice but was cleared from those of STAT1(+/+) mice without propagation. It was found that the anti-IFN capacity of the SeV C protein was indispensable for pathogenicity in mice. Conversely, the results show that the innate immunity contributed to elimination of SeV in early stages of infection in the absence of anti-IFN capacity.

Differential Receptor Subunit Affinities of Type I Interferons Govern Differential Signal Activation

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses by binding to a shared cell surface receptor comprising the transmembrane proteins ifnar1 and ifnar2. Activation of differential response patterns by IFNs has been observed, suggesting that members of the family play different roles in innate immunity. The molecular basis for differential signaling has not been identified yet. Here, we have investigated the recognition of various IFNs including several human IFNα species, human IFNω and human IFNβ as well as ovine IFNτ2 by the receptor subunits in detail. Binding to the extracellular domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC) was monitored in real time by reflectance interference and total internal reflection fluorescence spectroscopy. For all IFNs investigated, competitive 1:1 interaction not only with ifnar2-EC but also with ifnar1-EC was shown. Furthermore, ternary complex formation was studied with ifnar1-EC and ifnar2-EC tethered onto solid-supported membranes. These analyses confirmed that the signaling complexes recruited by IFNs have very similar architectures. However, differences in rate and affinity constants over several orders of magnitude were observed for both the interactions with ifnar1-EC and ifnar2-EC. These data were correlated with the potencies of ISGF3 activation, antiviral and anti-proliferative activity on 2fTGH cells. The ISGF3 formation and antiviral activity correlated very well with the binding affinity towards ifnar2. In contrast, the affinity towards ifnar1 played a key role for antiproliferative activity. A striking correlation was observed for relative binding affinities towards ifnar1 and ifnar2 with the differential antiproliferative potency. This correlation was confirmed by systematically engineering IFNα2 mutants with very high differential antiproliferative potency.

Activation of Tyk2 and Stat3 Is Required for the Apoptotic Actions of Interferon-β in Primary Pro-B Cells

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.

Interferonα Activates NF-κB in JAK1-deficient Cells through aTYK2-dependentPathway

In addition to activating members of the STAT transcription factor family, interferon alpha/beta (IFNalpha/beta) activates the NF-kappaB transcription factor. To determine the role of the Janus tyrosine kinase (JAK)-STAT pathway in NF-kappaB activation by IFN, we examined NF-kappaB activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH), IFN activates STAT1, STAT2, and STAT3, as well as NF-kappaB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells, IFN induces NF-kappaB activation and NF-kappaB dependent gene transcription but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-kappaB activation by IFN. Moreover, IFN does not promote NF-kappaB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-kappaB signaling pathway. In the IFN signaling pathway leading to STAT activation, both JAK1 and TYK2 are essential, whereas NF-kappaB activation requires only TYK2.

Functional Relevance of the Conserved DNA-binding Domain of STAT2

Several distinct type I interferon (IFN)-inducible STAT2-containing complexes have been identified. For the IFN-stimulated gene factor 3 (ISGF3), STAT1 and IRF-9 mediate IFN-stimulated response element (ISRE) binding, whereas STAT2 provides a potent transactivational domain. ISGF3-independent STAT2-containing complexes, specifically STAT2:1 and STAT2:3, bind a gamma-activated sequence (GAS)-like element, yet the contribution of each STAT to DNA binding is unknown. Moreover, the contribution of these ISGF3-independent STAT2-containing complexes to IFN-inducible responses is not defined. Accordingly, we generated mutant cDNAs, targeting the DNA-binding domain in STAT2. These cDNAs were introduced by transfection into U6A cells lacking STAT2, resulting in a panel of cell lines expressing mutant STAT2 proteins. Studies assessed the sensitivity of U6A cells reconstituted with intact STAT2 (U6A-2) and cells expressing mutant STAT2s (U6A-2E426A,E427A (EE-AA), U6A-2V453I, U6A-2V454I, U6A-2V454A, U6A-2V453I,V454I(VV-II), U6A-2N458A) to IFN-inducible responses. Our data reveal that none of the mutations in the STAT2 DNA-binding domain affected IFN-inducible ISGF3 activation, and only the VV-II mutation restricted antiviral and growth inhibitory responses to IFN. Indeed, U6A-2VV-II cells are refractory to these IFN-inducible biological activities and also exhibit impaired IFN-inducible GAS-driven transcriptional activation and subsequent gene expression. Chromatin immunoprecipitation assays revealed that the VV-II mutation in STAT2 does not abrogate, but reduces the DNA binding activity of STAT2:1 heterodimers. Taken together, these data suggest a role for the conserved DNA-binding domain of STAT2 specific to the activity of ISGF3-independent STAT2-containing complexes.

A Novel Method for Screening Viral Interferon-Resistance Genes

Many viruses have evolved mechanisms to antagonize the interferon (IFN) system, targeting all the major components involved in receptor binding and signaling. Although a number of these vital proteins are homologous to cellular proteins involved in IFN downregulation (e.g., viral IFN regulatory factors [vIRFs]), many share little resemblance to known proteins. To determine the IFN-blocking properties of these proteins, functional assays are required. Here, we present a new and rapid functional screening method, based on the 2fTGH cell line, which is able to determine viral gene products that inhibit the IFN-alpha/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-alpha and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-alpha. We also demonstrate that selection occurs if members of the Jak-Stat signaling pathway are lost. To show the utility of our system, we have used a known suppressor of IFN signaling, the human papillomavirus (HPV) E7 gene. Expression of E7 causes the loss of ability of 2fTGH cells to respond to IFN-alpha treatment because of a functional disruption of the signaling pathway. This approach offers a new strategy for identifying novel viral genes or new functions of already described viral genes that have a role in IFN-alpha signaling inhibition.

Identification of Genes in the Ovine Endometrium Regulated by Interferon τ Independent of Signal Transducer and Activator of Transcription 1

Interferon tau (IFNtau), a type I IFN produced by the conceptus trophectoderm, increases many type I IFN-stimulated genes (ISGs) in the ovine uterine endometrial stroma and glandular epithelium (GE) using signal transducer and activator of transcription 1 (STAT1)-dependent pathways. Most ISGs are not induced or increased by IFNtau in the STAT1-negative endometrial luminal epithelium (LE). The objective was to identify genes regulated by IFNtau in a STAT1-independent manner using DNA microarray and human cell lines. The RNA from human 2fTGH and U3A (STAT1 null 2fTGH) cell lines, stimulated for 24 h with nothing or recombinant ovine IFNtau, was profiled using an Affymetrix human genome U95Av2 microarray. In 2fTGH cells, IFNtau increased the expression of 101 genes at least 2-fold, including IFN-inducible 56-kDa protein (IFI56), ISG12 or p27, and guanylate binding protein isoform I (GBP-2). In U3A cells, IFNtau increased expression of 66 genes at least 2-fold, including Wnt7a. Steady-state levels of IFI56, ISG12, GBP-2, and Wnt7a mRNAs increased in the ovine uterine endometrium between d 10 and 16 of pregnancy but not during the estrous cycle. GBP-2 and IFI56 mRNAs were expressed only in endometrial stroma, ISG12 in both LE and GE, and Wnt7a only in LE of the ovine uterus. Intrauterine infusion of ovine IFNtau increased expression of all four genes in the endometrium of cyclic ewes. Therefore, IFNtau does regulate genes independent of STAT1 in the endometrial LE and U3A cells and dependent on STAT1 in the endometrial stroma and 2fTGH cells. These IFNtau -stimulated genes may be important in establishment of uterine receptivity to the embryo and conceptus implantation given their stage-specificity in endometrium across diverse species.