Type 1 interferon auto-antibodies are elevated in patients with decompensated liver cirrhosis.

Patients with decompensated liver cirrhosis, in particular those classified as Childs-Pugh class C, are at increased risk of severe coronavirus disease-2019 (COVID-19) upon infection with severe acute respiratory coronavirus 2 (SARS-CoV-2). The biological mechanisms underlying this are unknown. We aimed to examine the levels of serum intrinsic antiviral proteins as well as alterations in the innate antiviral immune response in patients with decompensated liver cirrhosis. Serum from 53 SARS-CoV-2 unexposed and unvaccinated individuals, with decompensated liver cirrhosis undergoing assessment for liver transplantation, were screened using SARS-CoV-2 pseudoparticle and SARS-CoV-2 virus assays. The ability of serum to inhibit interferon (IFN) signalling was assessed using a cell-based reporter assay. Severity of liver disease was assessed using two clinical scoring systems, the Child-Pugh class and the MELD-Na score. In the presence of serum from SARS-CoV-2 unexposed patients with decompensated liver cirrhosis there was no association between SARS-CoV-2 pseudoparticle infection or live SARS-CoV-2 virus infection and severity of liver disease. Type I IFNs are a key component of the innate antiviral response. Serum from patients with decompensated liver cirrhosis contained elevated levels of auto-antibodies capable of binding IFN-α2b compared to healthy controls. High MELD-Na scores were associated with the ability of these auto-antibodies to neutralize type I IFN signalling by IFN-α2b but not IFN-ß1a. Our results demonstrate that neutralizing auto-antibodies targeting IFN-α2b are increased in patients with high MELD-Na scores. The presence of neutralizing type I IFN-specific auto-antibodies may increase the likelihood of viral infections, including severe COVID-19, in patients with decompensated liver cirrhosis.

Point of care detection of SARS-CoV-2 antibodies and neutralisation capacity-lateral flow immunoassay evaluation compared to commercial assay to inform potential role in therapeutic and surveillance practices.

Introduction: As the COVID-19 pandemic moves towards endemic status, testing strategies are being de-escalated. A rapid and effective point of care test (POCT) assessment of SARS-CoV-2 immune responses can inform clinical decision-making and epidemiological monitoring of the disease. This cross-sectional seroprevalence study of anti-SARS-CoV-2 antibodies in Irish healthcare workers assessed how rapid anti-SARS-CoV-2 antibody testing can be compared to a standard laboratory assay, discusses its effectiveness in neutralisation assessment and its uses into the future of the pandemic. Methods: A point of care lateral flow immunoassay (LFA) detecting anti-SARS-CoV-2 spike (S)-receptor binding domain (RBD) neutralising antibodies (Healgen SARS-CoV-2 neutralising Antibody Rapid Test Cassette) was compared to the Roche Elecsys/-S anti-SARS-CoV-2 antibody assays and an in vitro surrogate neutralisation assay. A correlation between anti-spike (S), anti-nucleocapsid (N) titres, and in vitro neutralisation was also assessed. Results: 1,777 serology samples were tested using Roche Elecsys/-S anti-SARS-CoV-2 assays to detect total anti-N/S antibodies. 1,562 samples were tested using the POC LFA (including 50 negative controls), and 90 samples were tested using an in vitro ACE2-RBD binding inhibition surrogate neutralisation assay. The POCT demonstrated 97.7% sensitivity, 100% specificity, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 61% in comparison to the commercial assay. Anti-S antibody titres determined by the Roche assay stratified by the POC LFA result groups demonstrated statistically significant differences between the "Positive" and "Negative" LFA groups (p < 0.0001) and the "Weak Positive" and "Positive" LFA groups (p < 0.0001). No statistically significant difference in ACE2-RBD binding inhibition was demonstrated when stratified by the LFA POC results. A positive, statistically significant correlation was demonstrated between the in vitro pseudo-neutralisation assay results and anti-S antibody titres (rho 0.423, p < 0.001) and anti-N antibody titres (rho = 0.55, p < 0.0001). Conclusion: High sensitivity, specificity, and PPV were demonstrated for the POC LFA for the detection of anti-S-RBD antibodies in comparison to the commercial assay. The LFA was not a reliable determinant of the neutralisation capacity of identified antibodies. POC LFA are useful tools in sero-epidemiology settings, pandemic preparedness and may act as supportive tools in treatment decisions through the rapid identification of anti-Spike antibodies.

Optimal LC-MS metabolomic profiling reveals emergent changes to monocyte metabolism in response to lipopolysaccharide.

Introduction: Immunometabolism examines the links between immune cell function and metabolism. Dysregulation of immune cell metabolism is now an established feature of innate immune cell activation. Advances in liquid chromatography mass spectrometry (LC-MS) technologies have allowed discovery of unique insights into cellular metabolomics. Here we have studied and compared different sample preparation techniques and data normalisation methods described in the literature when applied to metabolomic profiling of human monocytes. Methods: Primary monocytes stimulated with lipopolysaccharide (LPS) for four hours was used as a study model. Monocytes (n=24) were freshly isolated from whole blood and stimulated for four hours with lipopolysaccharide (LPS). A methanol-based extraction protocol was developed and metabolomic profiling carried out using a Hydrophilic Interaction Liquid Chromatography (HILIC) LC-MS method. Data analysis pipelines used both targeted and untargeted approaches, and over 40 different data normalisation techniques to account for technical and biological variation were examined. Cytokine levels in supernatants were measured by ELISA. Results: This method provided broad coverage of the monocyte metabolome. The most efficient and consistent normalisation method was measurement of residual protein in the metabolite fraction, which was further validated and optimised using a commercial kit. Alterations to the monocyte metabolome in response to LPS can be detected as early as four hours post stimulation. Broad and profound changes in monocyte metabolism were seen, in line with increased cytokine production. Elevated levels of amino acids and Krebs cycle metabolites were noted and decreases in aspartate and ß-alanine are also reported for the first time. In the untargeted analysis, 154 metabolite entities were significantly altered compared to unstimulated cells. Pathway analysis revealed the most prominent changes occurred to (phospho-) inositol metabolism, glycolysis, and the pentose phosphate pathway. Discussion: These data report the emergent changes to monocyte metabolism in response to LPS, in line with reports from later time points. A number of these metabolites are reported to alter inflammatory gene expression, which may facilitate the increases in cytokine production. Further validation is needed to confirm the link between metabolic activation and upregulation of inflammatory responses.

Molluscum Contagiosum Virus Protein MC008 Targets NF-κB Activation by Inhibiting Ubiquitination of NEMO.

Molluscum contagiosum virus (MCV) is a human-adapted poxvirus that causes a common and persistent yet mild infection characterized by distinct, contagious, papular skin lesions. These lesions are notable for having little or no inflammation associated with them and can persist for long periods without an effective clearance response from the host. Like all poxviruses, MCV encodes potent immunosuppressive proteins that perturb innate immune pathways involved in virus sensing, the interferon response, and inflammation, which collectively orchestrate antiviral immunity and clearance, with several of these pathways converging at common signaling nodes. One such node is the regulator of canonical nuclear factor kappa B (NF-κB) activation, NF-κB essential modulator (NEMO). Here, we report that the MCV protein MC008 specifically inhibits NF-κB through its interaction with NEMO, disrupting its early ubiquitin-mediated activation and subsequent downstream signaling. MC008 is the third NEMO-targeting inhibitor to be described in MCV to date, with each inhibiting NEMO activation in distinct ways, highlighting strong selective pressure to evolve multiple ways of disabling this key signaling protein. IMPORTANCE Inflammation lies at the heart of most human diseases. Understanding the pathways that drive this response is the key to new anti-inflammatory therapies. Viruses evolve to target inflammation; thus, understanding how they do this reveals how inflammation is controlled and, potentially, how to disable it when it drives disease. Molluscum contagiosum virus (MCV) has specifically evolved to infect humans and displays an unprecedented ability to suppress inflammation in our tissue. We have identified a novel inhibitor of human innate signaling from MCV, MC008, which targets NEMO, a core regulator of proinflammatory signaling. Furthermore, MC008 appears to inhibit early ubiquitination, thus interrupting later events in NEMO activation, thereby validating current models of IκB kinase (IKK) complex regulation.

SARS-CoV-2 epidemiology, antibody dynamics, and neutralisation capacity in Irish healthcare workers in the era of booster COVID-19 vaccinations.

Background: The PRECISE Study, a multi-phase cross-sectional seroprevalence study of anti-SARS-CoV-2 antibodies in Irish healthcare workers (HCW) investigated: (1) risk factors for SARS-CoV-2 seropositivity, (2) the durability of antibody responses in a highly vaccinated HCW cohort, and (3) the neutralisation capacity of detected antibodies, prior to booster COVID-19 vaccination. Materials and methods: Serology samples were collected across two hospital sites in November 2021 and analysed using the Roche Elecsys Anti-SARS-CoV-2/Elecsys-S Anti-SARS-CoV-2 assays to detect anti-nucleocapsid (N) and anti-spike (S) antibodies respectively. Paired serology results from prior study phases were used to analyse changes in individual HCW serostatus over time. Risk-factors for SARS-CoV-2 infection were assessed for demographic and work-related factors. Antibody neutralisation capacity was assessed in a subset of samples via an in vitro ACE2 binding enzyme-linked immunosorbent assay. Results: 2,344 HCW samples were analysed. Median age was 43 years (IQR 33-50) with 80.5% (n = 1,886) female participants. Irish (78.9%, n = 1,850) and Asian (12.3%, n = 288) were the most commonly reported ethnicities. Nursing/midwifery (39.3%, n = 922) was the most common job role. 97.7% of participants were fully vaccinated, with Pfizer (81.1%, n = 1,902) and AstraZeneca (16.1%, n = 377) the most common vaccines received. Seroprevalence for anti-SARS-CoV-2 antibodies indicating prior infection was 23.4%, of these 33.6% represented previously undiagnosed infections. All vaccinated participants demonstrated positive anti-S antibodies and in those with paired serology, no individual demonstrated loss of previously positive anti-S status below assay threshold for positivity. Interval loss of anti-N antibody positivity was demonstrated in 8.8% of previously positive participants with paired results. Risk factors for SARS-CoV-2 seropositivity suggestive of previous infection included age 18-29 years (aRR 1.50, 95% CI 1.19-1.90, p < 0.001), India as country of birth (aRR 1.35, 95% CI 1.01-1.73, p = 0.036), lower education level (aRR 1.35, 95% CI 1.11-1.66, p = 0.004) and HCA job role (aRR 2.12, 95% CI 1.51-2.95, p < 0.001). Antibody neutralisation varied significantly by anti-SARS-CoV-2 antibody status, with highest levels noted in those anti-N positive, in particular those with vaccination plus previous SARS-CoV-2 infection. Conclusion: All vaccinated HCWs maintained anti-S positivity prior to COVID-19 booster vaccination, however anti-N positivity was more dynamic over time. Antibody neutralisation capacity was highest in participants with COVID-19 vaccination plus prior SARS-CoV-2 infection.

Bifurcation of signalling in human innate immune pathways to NF-kB and IRF family activation.

The human innate immune response can be activated through a wide range of stimuli. This multi-faceted system can be triggered by a range of immunostimulants including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These stimuli drive intracellular signalling pathways that branch off downstream to activate several distinct transcription factors. The two most impactful of which in innate immune outcomes are the NF-κB and the IRF family members. Both transcription factor families play defining roles in driving inflammation as well as the antiviral response. Pathways leading to their simultaneous activation share common upstream components but eventually distinct regulators which directly facilitate their activation. This review will discuss the current state of knowledge about what is known about how these pathways bifurcate to activate NF-κB and IRF family members.

Regulation of IRF3 activation in human antiviral signaling pathways.

The interferon regulatory factor (IRF) family of transcription factors play a vital role in the human innate antiviral immune responses with production of interferons (IFNs) as a hallmark outcome of activation. In recent years, IRF3 has been considered a principal early regulator of type I IFNs (TI-IFNs) directly downstream of intracellular virus sensing. Despite decades of research on IRF-activating pathways, many questions remain on the regulation of IRF3 activation. The kinases IκB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1) and the scaffold proteins TRAF family member-associated NF-kappa-B activator (TANK), NF-kappa-B-activating kinase-associated protein 1 (NAP1) and TANK-binding kinase 1-binding protein 1 (TBKBP1)/similar to NAP1 TBK1 adaptor (SINTBAD) are believed to be core components of an IRF3-activation complex yet their contextual involvement and complex composition are still unclear. This review will give an overview of antiviral signaling pathways leading to the activation of IRF3 and discuss recent developments in our understanding of its proximal regulation.

Previous SARS-CoV-2 Infection, Age, and Frailty Are Associated With 6-Month Vaccine-Induced Anti-Spike Antibody Titer in Nursing Home Residents.

OBJECTIVES: Older nursing home residents make up the population at greatest risk of morbidity and mortality from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. No studies have examined the determinants of long-term antibody responses post vaccination in this group. DESIGN: Longitudinal cohort study. SETTING AND PARTICIPANTS: Residents from 5 nursing homes assessed before vaccination, and 5 weeks and 6 months post vaccination, with the BNT162b2 messenger RNA SARS-CoV-2 vaccine. METHODS: Comprehensive clinical assessment was performed, including assessment for comorbidity, frailty, and SARS-CoV-2 infection history. Serum nucleocapsid and anti-spike receptor binding domain (RBD) antibodies were analyzed at all timepoints. An in vitro angiotensin-converting enzyme (ACE2) receptor-spike RBD neutralization assay assessed serum neutralization capacity. RESULTS: Of 86 participants (81.1 ± 10.8 years; 65% female), just under half (45.4%; 39 of 86) had evidence of previous SARS-CoV-2 infection. All participants demonstrated a significant antibody response to vaccination at 5 weeks and a significant decline in this response by 6 months. SARS-CoV-2 infection history was the strongest predictor of antibody titer (log-transformed) at both 5 weeks [ß: 3.00; 95% confidence interval (CI): 2.32-3.70; P < .001] and 6 months (ß: 3.59; 95% CI: 2.89-4.28; P < .001). Independent of SARS-CoV-2 infection history, both age in years (ß: -0.05; 95% CI: -0.08 to -0.02; P < .001) and frailty (ß: -0.22; 95% CI: -0.33 to -0.11; P < .001) were associated with a significantly lower antibody titer at 6 months. Anti-spike antibody titers at both 5 weeks and 6 months significantly correlated with in vitro neutralization capacity. CONCLUSIONS AND IMPLICATIONS: In older nursing home residents, SARS-CoV-2 infection history was the strongest predictor of anti-spike antibody titers at 6 months, whereas age and frailty were independently associated with lower titers at 6 months. Antibody titers significantly correlated with in vitro neutralization capacity. Although older SARS-CoV-2 naïve nursing home residents may be particularly vulnerable to breakthrough SARS-CoV-2 infection, the relationship between antibody titers, SARS-CoV-2 infection, and clinical outcomes remains to be fully elucidated in this vulnerable population.

Myeloid cell nuclear differentiation antigen controls the pathogen-stimulated type I interferon cascade in human monocytes by transcriptional regulation of IRF7.

Type I interferons (IFNs) are critical for anti-viral responses, and also drive autoimmunity when dysregulated. Upon viral sensing, monocytes elicit a sequential cascade of IFNß and IFNα production involving feedback amplification, but how exactly this cascade is regulated in human cells is incompletely understood. Here we show that the PYHIN protein myeloid cell nuclear differentiation antigen (MNDA) is required for IFNα induction in monocytes. Unlike other PYHINs, this is not due to a pathogen sensing role, but rather MNDA regulated expression of IRF7, a transcription factor essential for IFNα induction. Mechanistically, MNDA is required for recruitment of STAT2 and RNA polymerase II to the IRF7 gene promoter, and in fact MNDA is itself recruited to the IRF7 promoter after type I IFN stimulation. These data implicate MNDA as a critical regulator of the type I IFN cascade in human myeloid cells and reveal a new role for human PYHINs in innate immune gene induction.

Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity.

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera (n = 91) and pre-COVID-19 control sera (n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.

Poxviral Targeting of Interferon Regulatory Factor Activation.

As viruses have a capacity to rapidly evolve and continually alter the coding of their protein repertoires, host cells have evolved pathways to sense viruses through the one invariable feature common to all these pathogens-their nucleic acids. These genomic and transcriptional pathogen-associated molecular patterns (PAMPs) trigger the activation of germline-encoded anti-viral pattern recognition receptors (PRRs) that can distinguish viral nucleic acids from host forms by their localization and subtle differences in their chemistry. A wide range of transmembrane and cytosolic PRRs continually probe the intracellular environment for these viral PAMPs, activating pathways leading to the activation of anti-viral gene expression. The activation of Nuclear Factor Kappa B (NFκB) and Interferon (IFN) Regulatory Factor (IRF) family transcription factors are of central importance in driving pro-inflammatory and type-I interferon (TI-IFN) gene expression required to effectively restrict spread and trigger adaptive responses leading to clearance. Poxviruses evolve complex arrays of inhibitors which target these pathways at a variety of levels. This review will focus on how poxviruses target and inhibit PRR pathways leading to the activation of IRF family transcription factors.

Pathogenesis of ANCA-associated vasculitis: an emerging role for immunometabolism.

ANCA-associated vasculitis (AAV) is a severe systemic autoimmune disease. A key feature of AAV is the presence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) or proteinase-3 (PR3). ANCA are key to the pathogenesis of AAV, where they activate innate immune cells to drive inflammation. Pre-activation or 'priming' of immune cells appears to be important for complete cellular activation in AAV. The burgeoning field of immunometabolism has illuminated the governance of immune cell function by distinct metabolic pathways. There is ample evidence that the priming events synonymous with AAV alter immune cell metabolism. In this review we discuss the pathogenesis of AAV and its intersection with recent insights into immune cell metabolism.

Targeting of the cGAS-STING system by DNA viruses.

Innate sensing of viruses by cytosolic nucleic acid sensors is a key feature of anti-viral immunity against these pathogens. The DNA sensing pathway through the sensor cyclic GMP-AMP synthase (cGAS) and its downstream effector stimulator of interferon genes (STING) has emerged in recent years as a key, front-line means of driving interferons and pro-inflammatory cytokines in response to DNA virus infection in vertebrates. Unsurprisingly, many DNA viruses have evolved effective inhibitors of this signalling system which target at a wide variety of points from sensing all the way down to the activation of Interferon Regulatory Factor (IRF)-family and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-family transcription factors which drive a program of pro-inflammatory and anti-viral gene expression. Here we review DNA viruses that have been shown to inhibit this pathway and the inhibitors they have evolved to do it.

Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

Alkylating histone deacetylase inhibitors may have therapeutic value in experimental myeloperoxidase-ANCA vasculitis.

Current therapies for treating antineutrophil cytoplasm autoantibody (ANCA)-associated vasculitis include cyclophosphamide and corticosteroids. Unfortunately, these agents are associated with severe adverse effects, despite inducing remission in most patients. Histone deacetylase inhibitors are effective in rodent models of inflammation and act synergistically with many pharmacological agents, including alkylating agents like cyclophosphamide. EDO-S101 is an alkylating fusion histone deacetylase inhibitor molecule combining the DNA alkylating effect of Bendamustine with a pan-histone deacetylase inhibitor, Vorinostat. Here we studied the effects of EDO-S101 in two established rodent models of ANCA-associated vasculitis: a passive mouse model of anti-myeloperoxidase IgG-induced glomerulonephritis and an active rat model of myeloperoxidase-ANCA microscopic polyangiitis. Although pretreatment with EDO-S101 reduced circulating leukocytes, it did not prevent the development of passive IgG-induced glomerulonephritis in mice. On the other hand, treatment in rats significantly reduced glomerulonephritis and lung hemorrhage. EDO-S101 also significantly depleted rat B and T cells, and induced DNA damage and apoptosis in proliferating human B cells, suggesting a selective effect on the adaptive immune response. Thus, EDO-S101 may have a role in treatment of ANCA-associated vasculitis, operating primarily through its effects on the adaptive immune response to the autoantigen myeloperoxidase.

Molluscum Contagiosum Virus Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase Activation.

Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-κB activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-κB drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-κB activation. MC005 inhibited NF-κB proximal to the IκB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-κB essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions.IMPORTANCE Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-κB, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-κB by MC005 confirms current models of the complex way in which NF-κB is regulated and greatly expands our understanding of how MCV so effectively evades human immunity.

Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-B Activation by Targeting p65 for Degradation.

Molluscum contagiosum virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date, it is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus, but these are generally immunologically silent, suggesting the presence of potent inhibitors of human antiviral immunity and inflammation. Fewer than five MCV immunomodulatory genes have been characterized in detail, but it is likely that many more remain to be discovered given the density of such sequences in all well-characterized poxviruses. Following virus infection, NF-B activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines, such as tumor necrosis factor (TNF). As such, NF-B activation is required for virus detection, antiviral signaling, inflammation, and clearance of viral infection. Hence, we screened a library of MCV genes for effects on TNF-stimulated NF-B activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NF-B. Interestingly, MC132 also inhibited PRR- and virus-activated NF-B, since MC132 interacted with the NF-B subunit p65 and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NF-B p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity to persist in skin lesions.

Innate immune activation of NFκB and its antagonism by poxviruses.

In recent years there has been an acceleration of discovery in the field of innate anti-viral immunity to the point that many of the key events in early virus sensing and the discrete anti-viral responses they trigger have been elucidated in detail. In particular, pattern recognition receptors (PRRs) that detect viruses at the plasma membrane, in endosomes, and within the cytosol have been characterized. Upon stimulation by viruses, most of these PRRs trigger signal transduction pathways culminating in NFκB activation. NFκB contributes both to type I interferon induction, and to production of pro-inflammatory cytokines from infected cells. Our understanding of host anti-viral innate immunity has been greatly aided by an appreciation of the ways in which poxviruses have evolved strategies to inhibit both innate sensing and effector responses. A recurring feature of poxviral immunomodulation is the apparent necessity for poxviruses to evolve multiple, non-redundant inhibitors of NFκB activation which often appear to act on the same innate signalling pathway. The reason for such apparent over-targeting of one transcription factor is not clear. Here we describe the current understanding of how host cells sense poxvirus infection to trigger signalling pathways leading to NFκB activation and pro-inflammatory cytokine induction, and the ways in which poxviruses have evolved to concisely antagonize these systems.

Novel function of the unique N-terminal region of RUNX1c in B cell growth regulation.

RUNX family proteins are expressed from alternate promoters, giving rise to different N-terminal forms, but the functional difference of these isoforms is not understood. Here, we show that growth of a human B lymphoblastoid cell line infected with Epstein-Barr virus is inhibited by RUNX1c but not by RUNX1b. This gives a novel functional assay for the unique N-terminus of RUNX1c, and amino acids of RUNX1c required for the effect have been identified. Primary resting B cells contain RUNX1c, consistent with the growth inhibitory effect in B cells. The oncogene TEL-RUNX1 lacks the N-terminus of RUNX1c because of the TEL fusion and does not inhibit B cell growth. Mouse Runx1c lacks some of the sequences required for human RUNX1c to inhibit B cell growth, indicating that this aspect of human B cell growth control may differ in mice. Remarkably, a cell-penetrating peptide containing the N-terminal sequence of RUNX1c specifically antagonizes the growth inhibitory effect in B lymphoblastoid cells and might be used to modulate the function of human RUNX1c.