The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening ...studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prior studies have shown that 2'-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. ...Viruses lacking 2'-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2'-O methylation of the 5' viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1⁻/⁻ mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD(50) values in Ifit1⁻/⁻ compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1⁻/⁻ brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⁺ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2'-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A genetic absence of the common IFN-α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/-) mice is ...limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+) T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+) T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+) T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/-) mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+) T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+) T cell response at a stage distinct from the initial priming event.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are ...unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.
Viruses have evolved mechanisms of MHCI inhibition in order to evade recognition by cytotoxic CD8+ T cells (CTLs), which is well-illustrated by our prior studies on cowpox virus (CPXV) that encodes ...potent MHCI inhibitors. Deletion of CPXV viral MHCI inhibitors markedly attenuated in vivo infection due to effects on CTL effector function, not priming. However, the CTL response to CPXV in C57BL/6 mice is dominated by a single peptide antigen presented by H-2Kb. Here we evaluated the effect of viral MHCI inhibition on immunodominant (IDE) and subdominant epitopes (SDE) as this has not been thoroughly examined. We found that cross-priming, but not cross-dressing, is the main mechanism driving IDE and SDE CTL responses following CPXV infection. Secretion of the immunodominant antigen was not required for immunodominance. Instead, immunodominance was caused by CTL interference, known as immunodomination. Both immunodomination and cross-priming of SDEs were not affected by MHCI inhibition. SDE-specific CTLs were also capable of exerting immunodomination during primary and secondary responses, which was in part dependent on antigen abundance. Furthermore, CTL responses directed solely against SDEs protected against lethal CPXV infection, but only in the absence of the CPXV MHCI inhibitors. Thus, both SDE and IDE responses can contribute to protective immunity against poxviruses, implying that these principles apply to poxvirus-based vaccines.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent structural studies of the bacteriophage T7 DNA replication system have shed light on how multiple proteins assemble to copy two antiparallel DNA strands. In T7, acidic C-terminal tails of both ...the primase-helicase and single-stranded DNA binding protein bind to two basic patches on the DNA polymerase to aid in replisome assembly, processivity, and coordinated DNA synthesis. Although these electrostatic interactions are essential for DNA replication, the molecular details for how these tails bind the polymerase are unknown. We have determined an X-ray crystal structure of the T7 DNA polymerase bound to both a primer/template DNA and a peptide that mimics the C-terminal tail of the primase-helicase. The structure reveals that the essential C-terminal phenylalanine of the tail binds to a hydrophobic pocket that is surrounded by positive charge on the surface of the polymerase. We show that alterations of polymerase residues that engage the tail lead to defects in viral replication. In the structure, we also observe dTTP bound in the exonuclease active site and stacked against tryptophan 160. Using both primer/extension assays and high-throughput sequencing, we show how mutations in the exonuclease active site lead to defects in mismatch repair and an increase in the level of mutagenesis of the T7 genome. Finally, using small-angle X-ray scattering, we provide the first solution structures of a complex between the single-stranded DNA binding protein and the DNA polymerase and show how a single-stranded DNA binding protein dimer engages both one and two copies of DNA polymerase.
Although viral MHC class I inhibition is considered a classic immune-evasion strategy, its in vivo role is largely unclear. Mutant cowpox virus lacking its MHC class I inhibitors is markedly ...attenuated during acute infection because of CD8 ⁺ T-cell–dependent control, but it was not known how CD8 ⁺ T-cell responses are affected. Interestingly, we found no major effect of MHC class I down-regulation on priming of functional cowpox virus-specific CD8 ⁺ T cells. Instead, we demonstrate that, during acute infection in vivo, MHC class I down-regulation prevents primed virus-specific CD8 ⁺ T cells from recognizing infected cells and exerting effector responses to control the infection.
Phage Vardy is a lytic siphovirus isolated from creek soil in Cullowhee, NC, using Gordonia rubripertincta NRRL B-16540. Vardy’s 60,144-bp genome contains 90 predicted genes and five copies of a ...50-bp motif that may regulate gene expression. Based on gene content similarity, Vardy is assigned to cluster DJ.
Costimulation is required for optimal T cell activation, yet it is unclear whether poxviruses dedicatedly subvert costimulation during infection. Here, we report that the secreted M2 protein encoded ...by cowpox virus (CPXV) specifically interacts with human and murine B7.1 (CD80) and B7.2 (CD86). We also show that M2 competes with CD28 and CTLA4 for binding to cell surface B7 ligands, with stronger efficacy against CD28. Functionally, recombinant M2 and culture supernatants from wild-type (WT) but not M2-deficient (ΔM2) CPXV-infected cells can potently suppress B7 ligand-mediated T cell proliferation and interleukin-2 (IL-2) production. Furthermore, we observed increased antiviral CD4 and CD8 T cell responses in C57BL/6 mice challenged by ΔM2 CPXV compared with WT virus. These differences in immune responses to ΔM2 and WT CPXV were not observed in CD28-deficient mice. Taken together, our findings define a mechanism of viral sabotage of T cell activation that highlights the role of CD28 costimulation in host defense against poxvirus infections.
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