Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

ABSTRACT Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression. IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates

Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

Cytokine and growth factor signaling pathways involving STAT3 are frequently constitutively activated in many human primary tumors, and are known for the transcriptional role they play in controlling cell growth and cell cycle progression. However, the extent of STAT3's reach on transcriptional control of the genome as a whole remains an important question. We predicted that this persistent STAT3 signaling affects a wide variety of cellular functions, many of which still remain to be characterized. We took a broad approach to identify novel STAT3 regulated genes by examining changes in the genome-wide gene expression profile by microarray, using cells expressing constitutively-activated STAT3. Using computational analysis, we were able to define the gene expression profiles of cells containing activated STAT3 and identify candidate target genes with a wide range of biological functions. Among these genes we identified Necdin, a negative growth regulator, as a novel STAT3 target gene, whose expression is down-regulated at the mRNA and protein levels when STAT3 is constitutively active. This repression is STAT3 dependent, since inhibition of STAT3 using siRNA restores Necdin expression. A STAT3 DNA-binding site was identified in the Necdin promoter and both EMSA and chromatin immunoprecipitation confirm binding of STAT3 to this region. Necdin expression has previously been shown to be down-regulated in a melanoma and a drug-resistant ovarian cancer cell line. Further analysis of Necdin expression demonstrated repression in a STAT3-dependent manner in human melanoma, prostate and breast cancer cell lines. These results suggest that STAT3 coordinates expression of genes involved in multiple metabolic and biosynthetic pathways, integrating signals that lead to global transcriptional changes and oncogenesis. STAT3 may exert its oncogenic effect by up-regulating transcription of genes involved in promoting growth and proliferation, but also by down-regulating expression of negative regulators of the same cellular processes, such as Necdin

State of the world’s plants and fungi 2020

Kew’s State of the World’s Plants and Fungi project provides assessments of our current knowledge of the diversity of plants and fungi on Earth, the global threats that they face, and the policies to safeguard them. Produced in conjunction with an international scientific symposium, Kew’s State of the World’s Plants and Fungi sets an important international standard from which we can annually track trends in the global status of plant and fungal diversity

2-5A Antisense Treatment of Respiratory Syncytial Virus

Although a prominent cause of upper and lower respiratory tract disease in infants and the elderly, clinical options for treatment of respiratory syncytial virus (RSV) infections remain limited. Historically, attempts to develop vaccines have been unsuccessful, and rapid viral mutation rates have stifled development of several small molecule-based antiviral agents. Thus, targeted approaches to block RSV replication, including humanized monoclonal antibodies and nucleic acid-based strategies (antisense and RNA interference), have emerged as potentially viable drug development options

Functional Sites within the IHNV NonVirion Protein that Regulate Host Cellular Responses

Fish Rhabdoviruses are responsible for causing fatal epizootics within commercial and wild populations of various fish species around the world. Infectious hematopoietic necrosis virus (IHNV), also known as the Salmonid novirhabdovirus, is enzootic along the Pacific Coast of North America and is comprised of five genogroups, each of which is endemic to a specific geographical location. Once the virus enters the host through the fin epithelia, IHNV infection causes infectious hematopoietic necrosis in salmonid species. The disease is highly fatal and presents with signs such as abdominal distension, bulging of the eyes, anemia, and necrosis of vital organs such as the liver and kidneys, all caused by systemic hemorrhaging within the host. The 11-kb negative-sense, ssRNA viral genome within IHNV consists of six genes that encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), nonvirion protein (NV), and RNA-dependent RNA polymerase (L), in order from 3’ to 5’, respectively (Fig. 1). While most of the protein products from the IHNV genome have been studied and elucidated, the precise function of the NV protein remains unknown. While multiple studies have reported various roles for NV, such as suppression of apoptosis, interferon (IFN) induction, and NF-κB activation, data from our lab suggest that NV augments transcriptional or translational responses in the host. Using transient transfections and luciferase reporter assays, we have observed upregulation of host cell transcription/translation and innate immune responses. Regardless of the proposed functions of NV, functional sites within the viral protein are poorly defined. With the introduction of C- and N-terminal deletion mutations (∆NV), we were able to characterize the effects of mutated NV on rainbow trout gill epithelial cell (RT-Gill) constitutive and induced transcriptional responses using specific luciferase reporter plasmids, pCAGluc and RT-IFNluc. Our results suggest that while all ∆NV mutants showed a decrease in the augmented luciferase expression obtained with WT-NV, mutations within the N-terminal region of the protein led to an inhibitory effect on constitutive or induced luciferase expression. These data suggest that the N-terminal region of NV plays a critical role in the upregulation of host cell expression.https://corescholar.libraries.wright.edu/urop_celebration/1102/thumbnail.jp

Ectopic Expression of Toll-Like Receptor-3 (TLR-3) Overcomes the Double-Stranded RNA (dsRNA) Signaling Defects of P2.1 Cells

Cells respond to viral infection through induction of discrete, innate immune response pathways that lead to induction of interferons (IFNs) and other proinflammatory cytokines, as well as the direct induction of some IFN-responsive genes that mediate specific antiviral or immunomodulatory responses. To assess the classes of genes induced directly upon treatment of cells with double-stranded RNA (dsRNA), a mimic of viral infection, we made use of a mutant human cell line defective in responsiveness to dsRNA and IFN. P2.1 mutant cells were generated from a Jak1-minus, HT1080 fibrosarcoma-derived cell line (U4C) after extensive mutagenesis with the intercalating agent ICR191. We now demonstrate that P2.1 cells are defective in basal and induced expression of toll-like receptor-3 (TLR-3), which may contribute to their dsRNA-unresponsive phenotype. After transfection with a wild-type TLR-3 gene, P2.1 cells were largely responsive to a dsRNA challenge, as assessed by activation of NF-κB and IFN regulatory factors (IRFs) and induction of IFN-β and other genes. Untransfected and TLR-3-transfected P2.1 cells were assessed for global dsRNA responsiveness in oligonucleotide gene array studies alongside parental U4C and HT1080 cells. Several distinct patterns of gene induction in response to dsRNA challenge were identified, including genes expressed in a TLR-3-dependent manner, genes that required an intact IFN feedback for expression, and dsRNA-responsive genes that appeared not to require TLR-3 for induction. These data support the hypothesis that TLR-3 is an important determinant of cellular responses to external dsRNA and demonstrate distinctions in the repertoires of dsRNA-regulated genes induced when the IFN-feedback loop is present or absent in cells

Involvement of Noxa in Cellular Apoptotic Responses to Interferon, Double-stranded RNA, and Virus Infection

Double-stranded RNA (dsRNA) accumulates in virally infected cells, leading to induction of genes encoding proteins involved in signaling, apoptosis, protein synthesis/processing, and cell metabolism. Noxa is a BH3-containing mitochondrial protein that contributes to apoptosis by disrupting mitochondrial outer membrane integrity. Here we demonstrate potent induction of Noxa expression by exposure of cells to dsRNA, interferon (IFN), and virus. Noxa induction was confirmed by using reverse transcriptase-PCR and immunoblot analyses in multiple human tumor cell lines. Importantly, Noxa regulation by IFN and dsRNA was independent of p53, thereby identifying a novel mechanism of Noxa induction. Ectopic expression of Noxa in HT1080 fibrosarcoma cells enhanced cellular sensitivity to viral or dsRNA/actinomycin D-induced apoptosis, typified by enhanced cytochrome c release from the mitochondrial to the cytosolic fraction and increased cleavage of caspases 3 and 9. Point and deletion mutations of Noxa confirmed that both the BH3 domain and the mitochondrial-targeting domain were necessary for enhanced cellular apoptotic responses to dsRNA, IFN, or virus. Treatment of cells with dsRNA or virus, but not etoposide, induced interaction between Noxa and Bax that required an intact Noxa BH3 domain. Interestingly, the Noxa mitochondrial-targeting domain deletion mutant interacted with Bax in a dsRNA-dependent manner and redirected Bax away from the mitochondria, thus acting as a dominant-negative protein. Together, these data suggest that Noxa is an important component of the innate immune response of cells to viral infection, leading to enhanced cellular apoptosis that may play a role in limiting viral dissemination

Controlling Gene Expression with 2-5A Antisense

Recent work has demonstrated that the activity of a ubiquitous cellular enzyme, ribonuclease L (RNase L), can be harnessed to cleave targeted RNA species. Activation of RNase L is dependent on the presence of 2′,5′-linked oligoadenylates (2-5A), usually produced by cells infected with viruses. By conjugating synthetic 2-5A to specific antisense compounds, it is now possible to selectively degrade RNAs in an RNase L-dependent manner, thereby providing an alternative to RNase H-dependent approaches. In this summary, we provide an updated description of the synthesis procedure for constructing these chimeric 2-5A antisense molecules. Examples of successful applications of the 2-5A antisense strategy are described, along with some of the procedures involved in those studies. Several methods are also provided for optimizing compound uptake and analyzing their effects on cells. Finally, we discuss the current body of evidence that supports the contention that RNase L is indeed the primary mediator of 2-5A antisense effects and the possible implications that this has on the future of this therapeutic approach

Mitochondrial Localization and Pro-apoptotic Effects of the Interferon-inducible Protein ISG12a

ISG12a is one of the most highly induced genes following treatment of cells with type I interferons (IFNs). The encoded protein belongs to a family of poorly characterized, low molecular weight IFN-inducible proteins that includes 6–16 (G1P3), 1–8U (IFITM3), and 1–8D (IFITM2). Our studies demonstrate that the ISG12a protein associates with or inserts into the mitochondrial membrane. Transient expression of ISG12a led to decreased viable cell numbers and enhanced sensitivity to DNA-damage induced apoptosis, effects that were blocked by Bcl-2 co-expression or treatment with a pan-caspase inhibitor. ISG12a enhanced etoposide induced cytochrome c release, Bax activation and loss of mitochondrial membrane potential. siRNA-mediated inhibition of ectopic ISG12a protein expression prevented the sensitization to etoposide-induced apoptosis and also decreased the ability of IFN-β pretreatment to sensitize cells to etoposide, thereby demonstrating a role for ISG12a in this process. These data suggest that ISG12a contributes to IFN-dependent perturbation of normal mitochondrial function, thus adding ISG12a to a growing list of IFN-induced proteins that impact cellular apoptosis

Genes for the Trophoblast Interferons in Sheep, Goat and Musk Ox, and Distribution of Related Genes among Mammals

The trophoblast interferons (IFNs) are a family of Type 1 IFN found in domestic ruminants that are most closely related to the little-studied 172-amino-acid IFN-omega. They are produced in massive amounts by the preimplantation conceptus at a time coincident with maternal recognition of pregnancy, and are implicated in playing an important role in this process. Here we report the characterization of four distinctmembers of the ovine trophoblast IFN (oTP-1) gene family, and demonstrate that they, along with previously characterized bovine trophoblast (bTP-1) genes, possess distinctive promoter sequences when compared to ovine and bovine IFN-omega genes. Genomic Southern blot analysis of numerous mammalian species (zoo blots) indicate that, whereas the IFN-omega are widely distributed among mammals, genes for the trophoblast IFN appear to be limited to ruminant species within the Artiodactyla order. Further polymerase chain reaction (PCR) analysis of trophoblast IFN genes in these ruminant species has permitted isolation of genes for goat and musk ox trophoblast IFN. These data suggest that the trophoblast IFNs are a distinct family of IFN with a limited distribution among mammals. Read les