Distinct temporal regulation of intrinsic and innate intracellular immunity to Herpes Simplex Virus type 1 (HSV-1) infection

QR355 Virology

Alandijany, Thamir Abdulaziz A. ; Medical Research Council (MRC) ; Boutell, Chris

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: Intrinsic immunity, innate immunity, herpes, HSV-1, PML, IFI16, interferon, interferon stimulated genes, intracellular immunity, click chemistry.;   

Others  :  http://theses.gla.ac.uk/30662/7/2018AlandijanyPhD.pdf

来源: University of Glasgow

【 摘 要 】

Intrinsic and innate immunity play pivotal roles in limiting the replication of invading viral pathogens. Intrinsic immunity is constitutive and mediated by pre-existing host cell restriction factors (e.g., promyelocytic leukemia-nuclear body (PML-NB) constituent proteins) which directly confer antiviral properties. On the other hand, innate immunity is inducible and upregulated in response to infection. Pattern recognition receptors (PRRs) (e.g., interferon gamma inducible protein 16 (IFI16)) sense pathogen-associated molecular patterns (PAMPs) and induce downstream signaling cascades leading to the induction of Interferon-stimulated gene (ISG) products that confer antiviral properties. These two arms of immunity represent the first line of intracellular defense to HSV-1 infection. Indeed, rapid recruitment of intrinsic and innate immune factors to viral DNA (vDNA) has a significant bearing on the outcome of infection. However, the spatial and temporal regulation of this recruitment remains poorly defined due to the technical challenges associated with vDNA detection at multiplicities of infection (MOI) that do not saturate intrinsic host factors. Utilizing 5-Ethynyl-2’-deoxyuridine (EdU) labeling of HSV-1 DNA in combination with click chemistry, we directly visualized input viral genomes under low MOI conditions (MOI of ≤ 3 PFU/cell) at 30-90 minutes post-addition of virus (mpi). This protocol is sensitive, specific, and compatible with indirect immunofluorescence (IF) staining protocols, providing a valuable assay to investigate the temporal recruitment of immune regulators to infecting vDNA.Upon entry of vDNA into the nucleus, PML-NB associated restriction factors (e.g., PML, SP100, and Daxx) were rapidly recruited to infecting viral genome foci. This process occurred in a PML-dependent manner and led to genome entrapment and silencing within PML-NBs. Interestingly, genome entrapment was observed during both wild-type (WT) and ICP0-null mutant (ΔICP0) HSV-1 infection. During WT HSV-1 infection, ICP0 induced PML degradation and the dispersal of PML-NB restriction factors, highlighting the importance of ICP0 to release viral genomes entrapped within PML-NBs to stimulate the onset of lytic HSV-1 replication. During ΔICP0 HSV-1 infection, vDNA remained stably entrapped within PML-NBs leading to a repression in viral gene expression and a restriction in plaque formation. Importantly, IFI16 was not stably recruited to vDNA entrapped within PML-NBs, and ISG expression was not induced under low MOI conditions that do not saturate PML-NB intrinsic host defenses. These data demonstrate that vDNA entry into the nucleus alone is not sufficient to stimulate the induction of innate immunity.Saturation of intrinsic host defenses under high MOI conditions stimulated the stable recruitment of IFI16 to infecting viral genomes, and induced ISG expression in a PML-, IFI16-, and Janus-associated kinase (JAK)-dependent manner. The induction of this innate immune response was dependent on the onset of vDNA replication, as treatment of the infected cell monolayers with phosphonoacetic acid (PAA), a vDNA polymerase inhibitor, inhibited ISG induction in a dose-dependent manner. Unlike PML depletion, inhibition of JAK signaling failed to relieve the plaque formation defect of ΔICP0 HSV-1, but instead significantly enhanced virus yields.Collectively, these data, for the first time, demonstrate a temporal and sequential induction of intrinsic and innate immunity during HSV-1 infection. Intrinsic immunity is induced within minutes of nuclear infection to restrict the initiation of viral gene transcription and the onset of lytic replication. Escape from this intrinsic repression and initiation of vDNA replication, which takes several hours, triggers the induction of innate immunity. ISG products establish an antiviral state within infected and neighboring uninfected cells to constrict viral propagation and limit the spread of infection. We identify dual roles for PML in the regulation of intrinsic and innate immunity to HSV-1 infection. However, these host defenses are counteracted by the viral ubiquitin ligase ICP0, which targets PML for degradation to promote vDNA release from PML-NBs in order to evade intrinsic viral genome silencing from the onset of nuclear infection.