Cellular 5'-3' mRNA Exoribonuclease XRN1 Inhibits Interferon Beta Activation and Facilitates Influenza A Virus Replication.

Cellular 5'-3' exoribonuclease 1 (XRN1) is best known for its role as a decay factor, which by degrading 5' monophosphate RNA after the decapping of DCP2 in P-bodies (PBs) in Drosophila, yeast, and mammals. XRN1 has been shown to degrade host antiviral mRNAs following the influenza A virus (IAV) PA-X-mediated exonucleolytic cleavage processes. However, the mechanistic details of how XRN1 facilitates influenza A virus replication remain unclear. In this study, we discovered that XRN1 and nonstructural protein 1 (NS1) of IAV are directly associated and colocalize in the PBs. Moreover, XRN1 downregulation impaired viral replication while the viral titers were significantly increased in cells overexpressing XRN1, which suggest that XRN1 is a positive regulator in IAV life cycle. We further demonstrated that the IAV growth curve could be suppressed by adenosine 3',5'-bisphosphate (pAp) treatment, an inhibitor of XRN1. In virus-infected XRN1 knockout cells, the phosphorylated interferon regulatory factor 3 (p-IRF3) protein, interferon beta (IFN-ß) mRNA, and interferon-stimulated genes (ISGs) were significantly increased, resulting in the enhancement of the host innate immune response and suppression of viral protein production. Our data suggest a novel mechanism by which the IAV hijacks the cellular XRN1 to suppress the host innate immune response and to facilitate viral replication. IMPORTANCE A novel mechanistic discovery reveals that the host decay factor XRN1 contributes to influenza A virus replication, which exploits XRN1 activity to inhibit RIG-I-mediated innate immune response. Here, we identified a novel interaction between viral NS1 and host XRN1. Knockdown and knockout of XRN1 expression in human cell lines significantly decreased virus replication while boosting RIG-I-mediated interferon immune response, suggesting that XRN1 facilitates influenza A virus replication. The pAp effect as XRN1 inhibitor was evaluated; we found that pAp was capable of suppressing viral growth. To our knowledge, this study shows for the first time that a negative-strand and nucleus-replicating RNA virus, as influenza A virus, can hijack cellular XRN1 to suppress the host RIG-I-dependent innate immune response. These findings provide new insights suggesting that host XRN1 plays a positive role in influenza A virus replication and that the inhibitor pAp may be used in novel antiviral drug development.

Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein.

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14-NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.

The clinical phenotype of systemic sclerosis patients with anti-PM/Scl antibodies: results from the EUSTAR cohort.

OBJECTIVE: To evaluate clinical associations of anti-PM/Scl antibodies in patients with SSc in a multicentre international cohort, with particular focus on unresolved issues, including scleroderma renal crisis (RC), malignancies, and functional outcome of interstitial lung disease (ILD). METHODS: (1) Analysis of SSc patients from the EUSTAR database: 144 anti-PM/Scl+ without SSc-specific autoantibodies were compared with 7202 anti-PM/Scl-, and then to 155 anti-Pm/Scl+ with SSc-specific antibodies. (2) Case-control study: additional data were collected for 165 anti-PM/Scl+ SSc patients (85 from the EUSTAR registry) and compared with 257 anti-PM/Scl- SSc controls, matched for sex, cutaneous subset, disease duration and age at SSc onset. RESULTS: Patients with isolated anti-PM/Scl+, as compared with anti-Pm/Scl-, had higher frequency of muscle involvement, ILD, calcinosis and cutaneous signs of DM, but similar frequency of SRC and malignancies (either synchronous with SSc onset or not). The presence of muscle involvement was associated with a more severe disease phenotype. Although very frequent, ILD had a better functional outcome in cases than in controls. In patients with both anti-PM/Scl and SSc-specific antibodies, a higher frequency of typical SSc features than in those with isolated anti-PM/Scl was observed. CONCLUSION: The analysis of the largest series of anti-PM/Scl+ SSc patients so far reported helps to delineate a specific clinical subset with muscle involvement, cutaneous DM, calcinosis and ILD characterized by a good functional outcome. SRC and malignancies do not seem to be part of this syndrome.

The Relationship Between Autonomic Dysfunction of the Gastrointestinal Tract and Emotional Distress in Patients With Systemic Sclerosis.

BACKGROUND/OBJECTIVES: We hypothesized that emotional distress in systemic sclerosis (SSc) patients with moderate to severe gastrointestinal (GI) dysfunction is associated with dysautonomia. We sought to determine (1) the clinical characteristics associated with emotional distress in SSc, (2) the odds of having dysautonomia in those with emotional distress, and (3) whether GI dysautonomia, as measured by the Survey of Autonomic Symptoms (SAS), correlates with GI dysautonomia on the Composite Autonomic Symptom Score-31 (COMPASS-31). METHODS: Clinical and demographic features from our prospective cohort study were compared among SSc patients with and without GI-associated emotional distress (University of California at Los Angeles Scleroderma Clinical Trial Consortium Gastrointestinal Tract 2.0 well-being subscale >0.5 or ≤0.5) in cross-sectional analysis. Covariates/confounders independently associated with emotional distress were used to construct multivariable logistic regression models. The COMPASS-31 and SAS GI subdomains were compared with Spearman correlation. RESULTS: Forty-six patients with SSc were enrolled in the study. In univariate analyses, age (odds ratio [OR], 1.06; p = 0.026), severity of GI dysautonomia (COMPASS-31: OR, 1.41; p = 0.003), anti-centromere (A/B) antibodies (OR, 3.60; p = 0.044), and anti-PM-Scl (75/100) antibodies (OR, 0.15; p = 0.035) were associated with emotional distress. In the adjusted model, those with more severe GI dysautonomia remained more likely to have emotional distress (OR, 1.85; p = 0.026); those with anti-PM-Scl (75/100) antibodies were less likely to have emotional distress (OR, 0.03; p = 0.031). The SAS and COMPASS-31 GI subdomains moderately correlated (ρ = 0.68, p < 0.001). CONCLUSIONS: In SSc, increased symptom burden related to GI dysautonomia is associated with emotional distress. Multidisciplinary approaches addressing both the physical and emotional needs of the SSc patient may be warranted to optimize patient care.

Autoantibodies in outbred Swiss Webster mice following exposure to gold and mercury.

Exposure to heavy metals may have toxic effects on several human organs causing morbidity and mortality. Metals may trigger or exacerbate autoimmunity in humans. Inbred mouse strains with certain H-2 haplotypes are susceptible to xenobiotic-induced autoimmunity; and their immune response to metals such as mercury, gold, and silver have been explored. Serum antinuclear antibodies (ANA), polyclonal B-cell activation, hypergammaglobulinemia and tissue immune complex deposition are the main features of metal-induced autoimmunity in inbred mice. However, inbred mouse strains do not represent the genetic heterogeneity in humans. In this study, outbred Swiss Webster (SW) mice exposed to gold or mercury salts showed immune and autoimmune responses. Intramuscular injection of 22.5 mg/kg.bw aurothiomalate (AuTM) induced IgG ANA in SW mice starting after 5 weeks that persisted until week 15 although with a lower intensity. This was accompanied by elevated serum levels of total IgG antibodies against chromatin and total histones. Exposure to gold led to development of serum IgG autoantibodies corresponding to H1 and H2A histones, and dsDNA. Both gold and mercury induced polyclonal B-cell activation. Eight mg/L mercuric chloride (HgCl2) in drinking water, caused IgG antinucleolar antibodies (ANoA) after 5 weeks in SW mice accompanied by immune complex deposition in kidneys and spleen. Serum IgG antibodies corresponding to anti-fibrillarin, and anti-PM/Scl-100 antibodies, were observed in mercury-exposed SW mice. Gold and mercury trigger systemic autoimmune response in genetically heterogeneous outbred SW mice and suggest them as an appropriate model to study xenobiotic-induced autoimmunity.

The CCR4-NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression.

A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.

Performance evaluation of a line blot assay system for detection of anti-PM-Scl antibody in Japanese patients with systemic sclerosis.

OBJECTIVE: To evaluate the performance of a line blot (LB) assay for identifying anti-PM-Scl antibody (Ab). METHODS: We screened 56 serum samples from systemic sclerosis patients using a protein immunoprecipitation (IP) assay and an LB assay (Systemic Sclerosis Profile Euroline® Blot test kit) to detect anti-PM-Scl Ab. We compared the results obtained by the IP and LB assays. RESULTS: Among the 56 serum samples of SSc patients, 9 sera were positive for anti-PM-Scl75 Ab and 1 for anti-PM-Scl100 Ab by the LB assay. The protein IP assay revealed that none of the samples precipitated 75 or 100 kDa proteins identical to the anti-PM-Scl Ab reference serum, regardless of the positive or negative results obtained in the LB assay. Therefore, the false-positive rates for both anti-PM-Scl75 Ab and anti-PM-Scl100 Ab by the LB assay were 100%. CONCLUSION: Detection of anti-PM-Scl Ab assessed with a commercial LB assay requires extreme caution since it may yield false-positive data.

Clinical significance of autoantibodies in dermatomyositis and systemic sclerosis.

Autoimmune connective tissue diseases, including dermatomyositis and systemic sclerosis, have a heterogeneous clinical presentation and prognosis; moreover, their clinical features are often incomplete and overlap with other rheumatic disorders, which can make diagnosis and prognostic stratification challenging. Specific autoantibodies have been associated with certain clinical findings as well as prognostic implications, and many new associations have been made over the last decade. Although patient populations manifest considerable heterogeneity, autoantibodies can be used to help predict clinical features, prognosis, and response to therapy. In this review, the clinical and prognostic implications associated with disease-specific autoantibodies in dermatomyositis and scleroderma are summarized with an emphasis on how the clinician can use this information for patient care.

Calcinosis in poly-dermatomyositis: clinical and laboratory predictors and treatment options.

OBJECTIVES: We aimed to identify the possible clinical and laboratory predictors of calcinosis in a cohort of patients with a diagnosis of polymyositis (PM) and dermatomyositis (DM). METHODS: We carried out a retrospective analysis of a cohort of myositis patients attending our clinic between January 2013 and May 2014. RESULTS: 74 patients (58 females, 16 males) with PM (30 cases), DM (30 cases), overlap syndrome (13 cases) and inclusion body myositis (1 case) were enrolled. Sixteen patients (21.6%) had calcinosis that occurred a mean of 43.7 months after diagnosis of PDM. At multivariate analysis, patients with calcinosis experienced longer follow-up duration (p=0.006), anti-PM/Scl (p=0.033) and anti-NXP2 (p=0.024) positivity compared to patients without calcinosis. Furthermore, anti-NXP-2 positive C+ showed a diffuse form of calcinosis from the beginning and lower frequency of respiratory tract involvement. No single drug or associations of drugs was found effective in the treatment of calcinosis. CONCLUSIONS: A longer follow-up period of time, DM diagnosis and positivity for PM/Scl and NXP-2 could all be considered risk factors which foresee the development of calcinosis. Moreover, the positivity for antibodies to NXP-2 depicts a distinct phenotype of calcinosis with an early onset and quick widespread dissemination.

Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus.

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10-4) and 60% versus 28% (P = 3.10-8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10-10) and 82% (P = 5.10-13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.

Interpretation of an Extended Autoantibody Profile in a Well-Characterized Australian Systemic Sclerosis (Scleroderma) Cohort Using Principal Components Analysis.

OBJECTIVE: To determine the relationships between systemic sclerosis (SSc)-related autoantibodies, as well as their clinical associations, in a well-characterized Australian patient cohort. METHODS: Serum from 505 Australian SSc patients were analyzed with a commercial line immunoassay (EuroLine; Euroimmun) for autoantibodies to centromere proteins CENP-A and CENP-B, RNA polymerase III (RNAP III; epitopes 11 and 155), the 90-kd nucleolar protein NOR-90, fibrillarin, Th/To, PM/Scl-75, PM/Scl-100, Ku, topoisomerase I (topo I), tripartite motif-containing protein 21/Ro 52, and platelet-derived growth factor receptor. Patient subgroups were identified by hierarchical clustering of the first 2 dimensions of a principal components analysis of quantitative autoantibody scores. Results were compared with detailed clinical data. RESULTS: A total of 449 of the 505 patients were positive for at least 1 autoantibody by immunoblotting. Heatmap visualization of autoantibody scores, along with principal components analysis clustering, demonstrated strong, mutually exclusive relationships between CENP, RNAP III, and topo I. Five patient clusters were identified: CENP, RNAP III strong, RNAP III weak, topo I, and other. Clinical features associated with CENP, RNAP III, and topo I were consistent with previously published reports concerning limited cutaneous and diffuse cutaneous SSc. A novel finding was the statistical separation of RNAP III into 2 clusters. Patients in the RNAP III strong cluster had an increased risk of gastric antral vascular ectasia, but a lower risk of esophageal dysmotility. Patients in the other cluster were more likely to be male and to have a history of smoking and a history of malignancy, but were less likely to have telangiectasia, Raynaud's phenomenon, and joint contractures. CONCLUSION: Five major autoantibody clusters with specific clinical and serologic associations were identified in Australian SSc patients. Subclassification and disease stratification using autoantibodies may have clinical utility, particularly in early disease.

The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors.

2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.

Anti-SSA/Ro52 autoantibodies in scleroderma: results of an observational, cross-sectional study.

OBJECTIVES: To date, the diagnostic utility of anti-SSA/Ro52 autoantibodies in scleroderma and the association of them with certain clinical manifestations, particularly inflammatory myositis, are still controversial. This paper aims to assess the correlation between the presence of anti-SSA/Ro52 antibodies and the demographic, clinical and prognosis characteristics of patients with systemic sclerosis (SSc). METHODS: This is a retrospective, cross-sectional and observational study in patients with SSc. Baseline demographic and clinical characteristics were recorded. Presence of anti-SSA/Ro52, anti-SSA/Ro, anti-SSB/La, snRNP/Sm, anti-centromere, anti-Scl-70 and anti-PM-Scl were analysed by immunoblot, and antinuclear antibodies (ANA) by indirect immunofluorescence. Statistical analysis was performed with PASW Statics 18 software. RESULTS: A total of 132 consecutive patients with analysis of anti-SSA/Ro52 antibodies were selected from a Spanish cohort of 408 patients with SSc, 87.1% of them being women. About half of patients had the limited form (51.5%), followed by diffused form (18.9%), sclerosis sine scleroderma (22.7%), and pre-scleroderma (6.8%). Prevalence of anti-SSA/Ro52 was 35.6%. No association between anti-SSA/Ro52 and clinical manifestations was found, while detection of anti-SSA/Ro52 was significantly associated with the presence of anti-Ro. CONCLUSIONS: The results of our study show that anti-SSA/Ro52 antibodies are often found in SSc patients. No clinical manifestations, including inflammatory myopathy, were related with anti-SSA/Ro antibodies.

Clinical and serologic correlates of anti-PM/Scl antibodies in systemic sclerosis: a multicenter study of 763 patients.

OBJECTIVE: Anti-PM/Scl autoantibodies are found in polymyositis, dermatomyositis, systemic sclerosis (SSc), and systemic autoimmune disease overlap syndromes. PM-1α is a major epitope of the PM/Scl complex, and antibodies against PM-1α can be detected using a validated enzyme-linked immunosorbent assay (ELISA). This study was undertaken to determine the prevalence and identify the clinical correlates of anti-PM-1α antibodies in a large cohort of patients with SSc. METHODS: Serum samples were obtained from 763 patients with SSc enrolled in a multicenter Canadian cohort. The sera were analyzed by ELISA for the presence of antibodies against PM-1α. Associations between the presence of anti-PM-1α antibodies and demographic, clinical, and other serologic manifestations of SSc were investigated. RESULTS: Anti-PM-1α antibodies were present in 55 patients with SSc (7.2%), of whom almost 50% (26 of 55; 3.4% of the overall cohort) had no other SSc-specific antibodies, namely anticentromere, anti-topoisomerase I, and anti-RNA polymerase III. Features positively associated with the presence of anti-PM-1α antibodies included younger age at disease onset, skeletal muscle involvement, calcinosis, inflammatory arthritis, and overlap disease. Interstitial lung disease was less frequent and there were fewer gastrointestinal symptoms present in patients with anti-PM-1α antibodies compared to patients without these antibodies. CONCLUSION: Anti-PM-1α antibodies are relatively common in SSc and are associated with a distinct clinical phenotype, consistent with that described in association with other anti-PM/Scl autoantibodies. Although anti-PM-1α antibodies are not exclusive of other SSc-specific antibodies, they can be present in the absence thereof. Thus, anti-PM-1α antibodies may have considerable diagnostic and prognostic relevance in SSc.