Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.Wellcome-Trust, Lister Institute of Preventive Medicine U

Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA

Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV-1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid- and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo

Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity

Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-β also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture

Development and initial psychometric properties of the Warwick–Edinburgh Mental Wellbeing Scale-Intellectual Disability version

Background: The Warwick–Edinburgh Mental Wellbeing Scale (WEMWBS; Tennant et al., 2007) is yet to be validated in the intellectual disability (ID) population. The aim of this study was to report the development process and assess the psychometric properties of a newly adapted version of the WEMWBS and the Short WEMWBS for individuals with mild to moderate IDs (WEMWBS-ID/SWEMWBS-ID). / Method: The WEMWBS item wordings and response options were revised by clinicians and researchers expert in the field of ID, and a visual aid was added to the scale. The adapted version was reviewed by 10 individuals with IDs. The measure was administered by researchers online using screenshare, to individuals aged 16+ years with mild to moderate IDs. Data from three UK samples were collated to evaluate the WEMWBS-ID (n = 96). A subsample (n = 22) completed the measure again 1 to 2 weeks later to assess test–retest reliability, and 95 participants additionally completed an adapted version of the adapted Rosenberg Self-Esteem Scale to examine convergent validity. Additional data from a Canadian sample (n = 27) were used to evaluate the SWEMWBS-ID (n = 123). / Results: The WEMWBS-ID demonstrated good internal consistency (ω = 0.77–0.87), excellent test–retest reliability [intraclass correlation coefficient (ICC) =.88] and good convergent validity with the self-esteem scale (r =.48–.60) across samples. A confirmatory factor analysis for a single factor model demonstrated an adequate fit. The SWEMWBS-ID showed poor to good internal consistency (ω = 0.36–0.74), moderate test–retest reliability (ICC =.67) and good convergent validity (r =.48–.60) across samples, and a confirmatory factor analysis indicated good model fit for a single factor structure. / Conclusions: The WEMWBS-ID and short version demonstrated promising psychometric properties, when administered virtually by a researcher. Further exploration of the scales with larger, representative samples is warranted

DNA-PK Is Targeted by Multiple Vaccinia Virus Proteins to Inhibit DNA Sensing

Virus infection is sensed by pattern recognition receptors (PRRs) detecting virus nucleic acids and initiating an innate immune response. DNA-dependent protein kinase (DNA-PK) is a PRR that binds cytosolic DNA and is antagonized by vaccinia virus (VACV) protein C16. Here, VACV protein C4 is also shown to antagonize DNA-PK by binding to Ku and blocking Ku binding to DNA, leading to a reduced production of cytokines and chemokines in vivo and a diminished recruitment of inflammatory cells. C4 and C16 share redundancy in that a double deletion virus has reduced virulence not seen with single deletion viruses following intradermal infection. However, non-redundant functions exist because both single deletion viruses display attenuated virulence compared to wild-type VACV after intranasal infection. It is notable that VACV expresses two proteins to antagonize DNA-PK, but it is not known to target other DNA sensors, emphasizing the importance of this PRR in the response to infection in vivo

A mechanism for the inhibition of DNA-PK-mediated DNA sensing by a virus

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses