The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1

Rabies virus P protein inhibits alpha interferon (IFN-alpha)- and IFN-gamma-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-gamma and IFN-alpha, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-gamma- or IFN-alpha-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.     

TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

The nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small, excised damage-containing DNA oligonucleotides (sedDNAs) ∼30 nt in length. How cells process and degrade these byproducts of DNA repair is not known. Using a small scale RNA interference screen in UV-irradiated human cells, we identified TREX1 as a major regulator of sedDNA abundance. Knockdown of TREX1 increased the level of sedDNAs containing the two major UV photoproducts and their association with the NER proteins TFIIH and RPA. Overexpression of wild-type but not nuclease-inactive TREX1 significantly diminished sedDNA levels, and studies with purified recombinant TREX1 showed that the enzyme efficiently degrades DNA located 3′ of the UV photoproduct in the sedDNA. Knockdown or overexpression of TREX1 did not impact the overall rate of UV photoproduct removal from genomic DNA or cell survival, which indicates that TREX1 function in sedDNA degradation does not impact NER efficiency. Taken together, these results indicate a previously unknown role for TREX1 in promoting the degradation of the sedDNA products of the repair reaction. Because TREX1 mutations and inefficient DNA degradation impact inflammatory and immune signaling pathways, the regulation of sedDNA degradation by TREX1 may contribute to photosensitive skin disorders.     

T Cells Detect Intracellular DNA but Fail to Induce Type I IFN Responses: Implications for Restriction of HIV Replication

HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.     

Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome

TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.     

IFI16 protein mediates the anti-inflammatory actions of the type-I interferons through suppression of activation of caspase-1 by inflammasomes

Background

Type-I interferons (IFNs) are used to treat certain inflammatory diseases. Moreover, activation of type-I IFN-signaling in immune cells inhibits the production of proinflammatory cytokines and activation of inflammasomes. However, the molecular mechanisms remain largely unknown. Upon sensing cytosolic double-stranded DNA, the AIM2 protein forms the AIM2-ASC inflammasome, resulting in activation of caspase-1. Given that the IFI16 and AIM2 proteins are IFN-inducible and can heterodimerize with each other, we investigated the regulation of IFI16, AIM2, and inflammasome proteins by type-I and type-II IFNs and explored whether the IFI16 protein could negatively regulate the activation of the AIM2 (or other) inflammasome.

Methodology/ Principal Findings

We found that basal levels of the IFI16 and AIM2 proteins were relatively low in peripheral blood monocytes (CD14⁺) and in the THP-1 monocytic cell line. However, treatment of THP-1 cells with type-I (IFN-α or β) or type-II (IFN-γ) IFN induced the expression levels of IFI16, AIM2, ASC and CASP1 proteins. The induced levels of IFI16 and AIM2 proteins were detected primarily in the cytoplasm. Accordingly, relatively more IFI16 protein bound with the AIM2 protein in the cytoplasmic fraction. Notably, increased expression of IFI16 protein in transfected HEK-293 cells inhibited activation of caspase-1 by the AIM2-ASC inflammasome. Moreover, the constitutive knockdown of the IFI16 expression in THP-1 cells increased the basal and induced [induced by poly(dA:dT) or alum] activation of the caspase-1 by the AIM2 and NLRP3 inflammasomes.

Conclusions/Significance

Our observations revealed that the type-I and type-II IFNs induce the expression of IFI16, AIM2, and inflammasome proteins to various extents in THP-1 cells and the expression of IFI16 protein in THP-1 cells suppresses the activation of caspase-1 by the AIM2 and NLRP3 inflammasomes. Thus, our observations identify the IFI16 protein as a mediator of the anti-inflammatory actions of the type-I IFNs.     

Poxviral B1 kinase overcomes barrier to autointegration factor, a host defense against virus replication

Barrier to autointegration factor (BAF) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. Herein, we demonstrate a cytoplasmic role for BAF in host defense during poxviral infections. Vaccinia is the prototypic poxvirus, a family of DNA viruses that replicate exclusively in the cytoplasm of infected cells. Mutations in the vaccinia B1 kinase (B1) compromise viral DNA replication, but the mechanism by which B1 achieves this has remained elusive. We now show that BAF acts as a potent inhibitor of poxvirus replication unless its DNA-binding activity is blocked by B1-mediated phosphorylation. These data position BAF as the effector of an innate immune response that prevents replication of exogenous viral DNA in the cytoplasm. To enable the virus to evade this defense, the poxviral B1 has evolved to usurp a signaling pathway employed by the host cell.     

In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K(3) combination.

Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K(3) combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-microm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles.     

SRSF1 Facilitates Cytosolic DNA-Induced Production of Type I Interferons Recognized by RIG-I

BackgroundEvidence has shown that psoriasis is closely associated with infection; however, the mechanism of this association remains unclear. In mammalian cells, viral or bacterial infection is accompanied by the release of cytosolic DNA, which in turn triggers the production of type-I interferons (IFNs). Type I IFNs and their associated genes are significantly upregulated in psoriatic lesions. RIG-I is also highly upregulated in psoriatic lesions and is responsible for IFN production. However, RIG-I mediated regulatory signaling in psoriasis is poorly understood.MethodsWe screened a cDNA library and identified potential RIG-I interacting partners that may play a role in psoriasis.ResultsWe found that serine/arginine-rich splicing factor 1 (SRSF1) could specifically interact with RIG-I to facilitate RIG-I mediated production of type-I IFN that is triggered by cytosolic DNA. We found SRSF1 associates with RNA polymerase III and RIG-I in a DNA-dependent manner. In addition, treatment with a TNFα inhibitor downregulated SRSF1 expression in peripheral blood mononuclear cells (PBMCs) from psoriasis vulgaris patients.DiscussionBased on the abundance of pathogenic cytosolic DNA that is detected in psoriatic lesions, our finding that RIG-I interacts with SRSF1 to regulate type-I IFN production reveals a critical link regarding how cytosolic DNA specifically activates aberrant IFN expression. These data may provide new therapeutic targets for the treatment of psoriasis.     

TREX1 acts in degrading damaged DNA from drug-treated tumor cells

The major mammalian exonuclease TREX1 has been proposed to play a role in DNA repair and drug resistance. However, no cellular evidence substantiates this claim. Recent reports indicate TREX1's involvement in autoimmunity. To further understand its role, we studied TREX1 expression and functionality in anticancer drug-treated tumor cells. We report that the expression and localization of TREX1 are cell-type dependent. Camptothecin and other DNA damaging agents induced both TREX1 protein and its mRNA in a dose- and time-dependent manner. Using a TREX1-inducible cell line, we performed clonogenic assays and found no change in sensitivity of the cells to the agents upon TREX1 induction, suggesting that TREX1 may not play a role in DNA repair or drug sensitivity. Nevertheless, TREX1 serves as a key enzyme in the degradation of DNA from dying cells leading to less cellular DNA. Ubiquitously expressed in normal tissues, TREX1 may act in degrading DNA in all cell types undergoing a dying process before phagocytosis occurs.     

The TREX1 double-stranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3' --> 5' exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1WT/D200N and TREX1WT/D18N heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected approximately 2-fold lower rate than TREX1WT enzyme. Further, the D200N- and D18N-containing TREX1 homo- and heterodimers inhibit the dsDNA degradation activity of TREX1WT enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1R114H/R114H homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1WT enzyme, whereas the TREX1WT/R114H heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.     

Differential effects of CpG DNA on IFN-beta induction and STAT1 activation in murine macrophages versus dendritic cells: alternatively activated STAT1 negatively regulates TLR signaling in macrophages

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

The crystal structure of TREX1 explains the 3' nucleotide specificity and reveals a polyproline II helix for protein partnering

The TREX1 enzyme processes DNA ends as the major 3' --> 5' exonuclease activity in human cells. Mutations in the TREX1 gene are an underlying cause of the neurological brain disease Aicardi-Goutières syndrome implicating TREX1 dysfunction in an aberrant immune response. TREX1 action during apoptosis likely prevents autoimmune reaction to DNA that would otherwise persist. To understand the impact of TREX1 mutations identified in patients with Aicardi-Goutières syndrome on structure and activity we determined the x-ray crystal structure of the dimeric mouse TREX1 protein in substrate and product complexes containing single-stranded DNA and deoxyadenosine monophosphate, respectively. The structures show the specific interactions between the bound nucleotides and the residues lining the binding pocket of the 3' terminal nucleotide within the enzyme active site that account for specificity, and provide the molecular basis for understanding mutations that lead to disease. Three mutant forms of TREX1 protein identified in patients with Aicardi-Goutières syndrome were prepared and the measured activities show that these specific mutations reduce enzyme activity by 4-35,000-fold. The structure also reveals an 8-amino acid polyproline II helix within the TREX1 enzyme that suggests a mechanism for interactions of this exonuclease with other protein complexes.     

CYTOPLASMIC LABEL FOLLOWING TRITIATED THYMIDINE TREATMENT OF ALLIUM CEPA L. ROOTS : Cytochemical and Electron Microscope Study

Tritiated thymidine routinely labels onion root cytoplasm during most of the cell cycle. One-third of this label could be cytochemically identified as DNA. The balance of the label was not RNA or a lipid, or attributable to labeled impurities in thymidine-³H. In electron microscope radioautographs one-third of the cytoplasmic silver grains was over organelles, presumably mitochondria and plastids. The other two-thirds of the silver grains in electron micrographs was distributed widely, 41% over ground cytoplasm and 10% over cell walls-cell membranes. Snake venom phosphodiesterase (SVDase) extracted a cytoplasmic fraction not degraded by DNase, and did not appear to extract nuclear DNA. The SVDase-extractable fraction may be DNA or a thymidine 5'-phosphoryl group in an ester linkage with another hydroxylic compound. The nature of the nonextractable fraction is considered. Possibilities discussed are: (1) technical problems such as the binding of an acid-labile nuclear DNA in the cytoplasm; (2) non-DNA, such as breakdown products, and thymine compounds other than DNA; (3) DNA, not extractable because of the nature of its binding to other compounds or because it is a "core" resistant to DNase. Until the chemical nature of this nonextractable fraction is known, cytoplasmic label following thymidine-³H treatment cannot necessarily be considered DNA, nor the assumption made that thymidine-³H exclusively labels DNA.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

RIG-I mediates the co-induction of tumor necrosis factor and type I interferon elicited by myxoma virus in primary human macrophages

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.