Periodic Report Summary - DNA RECOGNITION (Recognition of viral DNA by the innate immune system) The objectives of the current project were to describe the role of cytosolic viral DNA in initiating the innate immune response to HSV and characterise the mechanism of DNA release from the virion into the cytosol.The aim of the project was to investigate the recognition of viral DNA by pattern recognition receptors (PRRs), to activate the innate immune response. Specifically, we aimed to identify cellular mechanisms of recognition of viral DNA and how this DNA is exposed for sensing.Prior to the commencement of this project, viral DNA sensors that had been identified were restricted to specific cells or specific DNA motifs. We have identified IFI16, a PYHIN family protein, as a novel DNA sensor, recognising HSV-1. Utilising immunofluorescence studies, we have demonstrated that IFI16 co-localises with transfected HSV-1 derived DNA and virally delivered HSV-1 DNA in the cytosol of THP1 macrophage cell line and primary human macrophages. This co-localisation precedes the recruitment of STING, an essential component of the DNA recognition signalling cascade, indicating that IFI16 recognition of DNA occurs prior to STING recruitment. In addition, utilising siRNA technology, we have shown that the murine ortholog of IFI16, p204 is required for optimal induction of IFN-beta, and pro-inflammatory cytokines detected in response to HSV-1 infection, determined by both qPCR and ELISA. These novel findings were recently published in Nature Immunology in a collaborative study with Andrew Bowie (Trinity College, Dublin) and Katherine Fitzgerald (University of Massachusetts). Furthermore we demonstrate that IFI16-dependent recognition of HSV-1 DNA is not restricted to a specific cell type. To clarify the nature of the pathogen-associated molecular pattern (PAMP), which is sensed to induce IFN-beta we investigated a series of HSV-1-derived sequences and demonstrated that the sequence was not of utmost importance, but the length of the sequence needed to be a minimum of 40bp.The release of viral DNA into the cytosol, were it is proposed to be recognised by cytosolic DNA sensors had not been conclusively demonstrated. Utilising Fluorescence in situ hybridisation (FISH) and confocal microscopy, the current work has demonstrated for the first time that HSV DNA is indeed visible in the cytosol of HSV infected cells at early time points following infection. DNA may gain access to the cytosol via a number of potential mechanisms such as autophagic degradation of the capsid, proteasomal degradation of the capsid or via uncoating of DNA in the nucleus and release via the nuclear export machinery. In addition, the route of entry may also contribute to the exposure of DNA to the cytosol, for instance DNA may be more likely to be exposed to the cytosol if the viral particle fuses directly with plasma membrane rather than if entry occurs via endocytosis, or vice versa. To determine the mechanism by which DNA gains access to the cytosol a number of inhibitors were used, to specifically inhibit the cellular functions which may contribute to the exposure of HSV-1 DNA to the cytosol. It was shown that the route of entry, autophagy and export from the nucleus were not mechanisms involved in the exposure of viral DNA to the cytosol. However, the exposure of DNA to the cytosol was strongly dependent on a functional proteasome. Consistent with this HSV-1 capsid co-localises with both ubiquitin, a modification often associated with targeting proteins for degradation, and the proteasome itself. Thus the current work indicates that HSV-1 DNA can indeed be exposed to the cytosol and that this occurs via the ubiquitination and subsequent proteasomal degradation of the viral capsid.Recent work by others has demonstrated that IFI16 also senses viral DNA in the nucleus and plays a role in the activation of the inflammasome to generate IL-1-beta. However, these reports suggest conflicting roles for IFI16 in different cell types. In addition, IFI16 restricts the life cycle of human cytomegalovirus, another herpesvirus family member. Finally, the role of IFI16 in the sensing of viral DNA has been proposed to be a secondary event downstream of another sensor, DDX41, in human monocytes, were IFI16 expression is very low. To clarify the role IFI16 in the innate immune response to herpesviruses, we generated stable knock-down of IFI16 in human macrophages, were we, in this project and others have demonstrated an abundance of IFI16 expression in un-stimulated/infected cells. Utilising this approach, the current project clearly demonstrated that IFI16 is an essential sentinel of cytoplasmic DNA inducing IFN-β, but not interleukin 1-beta, in response to infection with HSV-1, and CMV but not KSHV. Furthermore, we also demonstrated that IFI16 is a vital component of the sensing machinery responsible for the induction of IFN-beta following Listeria monocytogenes infection.The site of DNA was assumed to be the cytosol, however, IFI16 is a predominantly nuclear protein and this coupled with work by others demonstrating a role for IFI16 in sensing viral DNA in the nucleus, led us to further investigate the site at which viral DNA is recognised, leading to IFN-beta induction. Utilising chemical inhibition of nuclear export and a mutant HSV-1, unable to deliver it genome to the nucleus, we demonstrate in the current project that it is cytosolic viral DNA, not nuclear viral DNA, is the predominant activator of IFN-beta induction via IFI16.The current project demonstrates a clear step forward in our understanding of the mechanisms of innate immunity following viral and bacterial infection.
