Search

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated [...]

Nucleic acids are potent triggers for innate immunity. Double-stranded DNA and RNA adopt different helical conformations, including the unusual Z-conformation. Z- DNA/ RNA is recognised by Z-binding domains ( ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM-1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock-in models, we report that mutation of key amino acids involved in Z- DNA/ RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1- ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross-linked to endogenous RNA. These observations show that Z- RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1. [ABSTRACT FROM AUTHOR]

DNA-dependent activator of interferon regulatory factors/Z- DNA binding protein 1 ( DAI/ ZBP1) is a crucial sensor of necroptotic cell death induced by murine cytomegalovirus ( MCMV) in its natural host. Here, we show that viral capsid transport to the nucleus and subsequent viral IE3-dependent early transcription are required for necroptosis. Necroptosis induction does not depend on input virion DNA or newly synthesized viral DNA. A putative RNA-binding domain of DAI/ ZBP1, Zα2, is required to sense virus and trigger necroptosis. Thus, MCMV IE3-dependent transcription from the viral genome plays a crucial role in activating DAI/ ZBP1-dependent necroptosis. This implicates RNA transcripts generated by a large double-stranded DNA virus as a biologically relevant ligand for DAI/ ZBP1 during natural viral infection. [ABSTRACT FROM AUTHOR]

Murine gammaherpesvirus 68 (γHV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirely cleared by 2 weeks postinfection. Spontaneous reactivation of γHV68 from latently infected splenocytes upon ex vivo culture can readily be detected at the early stages of infection (e.g., day 16). However, by 6 weeks postinfection, very little spontaneous reactivation is detected upon explant into tissue culture. Here we report that stimulation of latently infected splenic B cells harvested at late times postinfection with cross-linking surface immunoglobulin (Ig), in conjunction with anti-CD40 antibody treatment, triggers virus reactivation. As expected, this treatment resulted in B-cell activation, as assessed by upregulation of CD69 on B cells, and ultimately B-cell proliferation. Since anti-Ig/anti-CD40 stimulation resulted in splenic B-cell proliferation, we assessed whether this reactivation stimulus could overcome the previously characterized defect in virus reactivation of a v-cyclin null γHV68 mutant. This analysis demonstrated that anti-Ig/anti-CD40 stimulation could drive reactivation of the v-cyclin null mutant virus in latently infected splenocytes, but not to the levels observed with wild-type γHV68. Thus, there appears to be a role for the v-cyclin in B cells following anti-Ig/ anti-CD40 stimulation independent of the induction of the cell cycle. Finally, to assess signals that are not mediated through the B-cell receptor, we demonstrate that addition of lipopolysaccharide to explanted splenocyte cultures also enhanced virus reactivation. These studies complement and extend previous analyses of Epstein-Barr virus and Kaposi's sarcoma-associated virus reactivation from latently infected cell lines by investigating reactivation of γHV68 from latently infected primary B cells recovered from infected hosts. [ABSTRACT FROM AUTHOR]

Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and β-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections. [ABSTRACT FROM AUTHOR]

Necroptosis initiated by the host sensor Z-NA Binding Protein-1 (ZBP1) is essential for host defense against a growing number of viruses, including Herpes Simplex Virus-1 (HSV-1). Studies with HSV-1 and other necroptogenic stimuli in murine settings have suggested that ZBP1 triggers necroptosis by directly complexing with the kinase RIPK3. Whether this is also the case in human cells, or whether additional co-factors are needed for ZBP1-mediated necroptosis, is unclear. Here, we show that ZBP1-induced necroptosis in human cells requires RIPK1. We have found that RIPK1 is essential for forming a stable and functional ZBP1-RIPK3 complex in human cells, but is dispensable for the formation of the equivalent murine complex. The RIP Homology Interaction Motif (RHIM) in RIPK3 is responsible for this difference between the two species, because replacing the RHIM in human RIPK3 with the RHIM from murine RIPK3 is sufficient to overcome the requirement for RIPK1 in human cells. These observations describe a critical mechanistic difference between mice and humans in how ZBP1 engages in necroptosis, with important implications for treating human diseases., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.