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      IFI16 Restricts HSV-1 Replication by Accumulating on the HSV-1 Genome, Repressing HSV-1 Gene Expression, and Directly or Indirectly Modulating Histone Modifications

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          Interferon-γ inducible factor 16 (IFI16) is a multifunctional nuclear protein involved in transcriptional regulation, induction of interferon-β (IFN-β), and activation of the inflammasome response. It interacts with the sugar-phosphate backbone of dsDNA and modulates viral and cellular transcription through largely undetermined mechanisms. IFI16 is a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus (HSV-1), though the mechanisms of HSV-1 restriction are not yet understood. Here, we show that IFI16 has a profound effect on HSV-1 replication in human foreskin fibroblasts, osteosarcoma cells, and breast epithelial cancer cells. IFI16 knockdown increased HSV-1 yield 6-fold and IFI16 overexpression reduced viral yield by over 5-fold. Importantly, HSV-1 gene expression, including the immediate early proteins, ICP0 and ICP4, the early proteins, ICP8 and TK, and the late proteins gB and Us11, was reduced in the presence of IFI16. Depletion of the inflammasome adaptor protein, ASC, or the IFN-inducing transcription factor, IRF-3, did not affect viral yield. ChIP studies demonstrated the presence of IFI16 bound to HSV-1 promoters in osteosarcoma (U2OS) cells and fibroblasts. Using CRISPR gene editing technology, we generated U2OS cells with permanent deletion of IFI16 protein expression. ChIP analysis of these cells and wild-type (wt) U2OS demonstrated increased association of RNA polymerase II, TATA binding protein (TBP) and Oct1 transcription factors with viral promoters in the absence of IFI16 at different times post infection. Although IFI16 did not alter the total histone occupancy at viral or cellular promoters, its absence promoted markers of active chromatin and decreased those of repressive chromatin with viral and cellular gene promoters. Collectively, these studies for the first time demonstrate that IFI16 prevents association of important transcriptional activators with wt HSV-1 promoters and suggest potential mechanisms of IFI16 restriction of wt HSV-1 replication and a direct or indirect role for IFI16 in histone modification.

          Author Summary

          HSV-1, a ubiquitous human pathogen that establishes a life-long infection, has evolved several mechanisms to evade host immune detection and responses. However, it is still subject to regulation by cellular factors. Recently, a host nuclear protein, IFI16, was shown to be involved in the innate defense response to HSV-1 infection. Here, we provide the first evidence that IFI16 inhibits wild-type HSV-1 replication by repressing viral gene expression independent of its roles in the immune response. We show that IFI16 binds the HSV-1 genome at the transcription start sites of several HSV-1 genes. Using a permanently IFI16-negative cell line that we generated, we demonstrate that IFI16 reduces the association of important transcription factors. IFI16 also promotes global histone modifications by increasing the markers of repressive chromatin and decreasing the markers for activating chromatin on viral and cellular genes. These insights into the role of IFI16 in HSV-1 biology suggest that stabilization of IFI16 is an attractive avenue for antiviral drug development.

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          Most cited references 101

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

           K Livak,  T Schmittgen (2001)
          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            RNA-guided human genome engineering via Cas9.

            Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
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              AIM2 recognizes cytosolic dsDNA and forms a caspase-1 activating inflammasome with ASC

              The innate immune system senses nucleic acids via germ-line encoded pattern recognition receptors. RNA is sensed via Toll-like receptor (TLR)−3, −7 and −8 or by the RNA helicases RIG-I and MDA-51. Little is known about sensors for cytoplasmic DNA which trigger antiviral and/or inflammatory responses2–6. The best characterized of these responses involves activation of the TANK-binding kinase (TBK1)-Interferon Regulatory Factor (IRF)-3 signaling axis to trigger transcriptional induction of IFN〈/® genes2,3. A second, less well-defined pathway leads to the activation of an ‘inflammasome’ which via caspase-1, controls the catalytic cleavage of the pro-forms of the cytokines IL-1β and IL-186,7. Here we identify the IFI20X/IFI16 (PYHIN) family member8, absent in melanoma 2 (AIM2), as a receptor for cytosolic DNA which regulates caspase-1. The HIN200 domain of AIM2 binds to DNA, while the PYD domain (but not that of the other PYHIN family members) associates with the adapter molecule ASC to activate both NF-κB and caspase-1. Knockdown of AIM2 abrogates caspase-1 activation in response to cytoplasmic dsDNA and the dsDNA virus, vaccinia. Collectively, these observations identify AIM2 as a novel receptor for cytoplasmic DNA, which forms an inflammasome with the ligand and ASC to activate caspase-1.

                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                November 2014
                6 November 2014
                : 10
                : 11

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Pages: 26
                This study was supported in part by the Public Health Service grants CA 180758, and the Rosalind Franklin University of Medicine and Science H. M. Bligh Cancer Research Fund to BC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Biology and Life Sciences
                Medical Microbiology
                Microbial Pathogens
                Viral Pathogens
                Herpes Simplex Virus
                Herpes Simplex Virus-1
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Gene Expression and Vector Techniques
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

                Infectious disease & Microbiology


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