The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis

IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond

Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

Summary: Foxp3+ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and “toxic” gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues. : An inflammatory environment threatens the stability of Foxp3+ Treg cells. Garg et al. show that by expressing the transcriptional regulator Blimp1, Treg cells resist the IL-6-driven loss of Foxp3 in inflamed tissues. Blimp1 prevents the methylation and reduced expression of Foxp3 through inhibition of the methyltransferase Dnmt3a. Keywords: regulatory T cells, Blimp1, CNS2, epigenetic regulation, CNS, inflammation, DNA methyltransferases, Foxp3, Interleukin-

Blocking IL-6 trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance

SummaryInterleukin-6 (IL-6) plays a paradoxical role in inflammation and metabolism. The pro-inflammatory effects of IL-6 are mediated via IL-6 “trans-signaling,” a process where the soluble form of the IL-6 receptor (sIL-6R) binds IL-6 and activates signaling in inflammatory cells that express the gp130 but not the IL-6 receptor. Here we show that trans-signaling recruits macrophages into adipose tissue (ATM). Moreover, blocking trans-signaling with soluble gp130Fc protein prevents high-fat diet (HFD)-induced ATM accumulation, but does not improve insulin action. Importantly, however, blockade of IL-6 trans-signaling, unlike complete ablation of IL-6 signaling, does not exacerbate obesity-induced weight gain, liver steatosis, or insulin resistance. Our data identify the sIL-6R as a critical chemotactic signal for ATM recruitment and suggest that selectively blocking IL-6 trans-signaling may be a more favorable treatment option for inflammatory diseases, compared with current treatments that completely block the action of IL-6 and negatively impact upon metabolic homeostasis

Evidence for novel hepaciviruses in rodents

CITATION: Drexler, J. F. et al. 2013. Evidence for novel hepaciviruses in rodents. PLoS Pathogens, 9(6): e1003438, doi:10.1371/journal.ppat.1003438.The original publication is available at http://journals.plos.org/plospathogensHepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1003438Publisher's versio

Presentation of hepacivirus infection in bank voles.

<p>A. Hepacivirus RNA concentrations in Myodes glareolus. Bars represent means and standard deviation of hepacivirus clade 1- and 2-positive organs and serum. The number of biological replicates is indicated below bars. B. In situ hybridization of rodent hepacivirus clade 1 RNA in M. glareolus. Viral RNA was stained in liver tissue of <i>M .glareolus</i> specimen RMU10-3379 (viral RNA concentration, 9.4×10⁸ copies per gram, GenBank accession no. KC411778). The RNA-negative specimen RMU10-3187 from the same species was processed identically and is shown below as a control. Positive staining is visible as distinct red granules in the cytoplasm of hepatocytes. Magnification was 100×, the inserts shows details of single hepatocytes in 10× higher magnification. Scale bars are shown to the lower right. C. Histopathology of M. glareolus liver specimens. Liver sections were stained by Hematoxylin and Eosin (H&E) and Epson van Giesson (EvG) stains. In H&E stains, black arrows point to inflammatory lymphocytic infiltrate. In EvG stains, black arrows highlight potential signs of fibrosis. Specimen 3180 shows intermediate portal inflammatory lymphocytic activity with potential low-grade fibrosis in a case with high hepacivirus RNA concentrations (1.5×10⁸ copies/gram). Specimen 1602 shows low-grade portal inflammatory activity and low-grade fibrosis in a case with high hepacivirus RNA concentrations (3.4×10⁸ copies/gram). Specimen 3187 shows no significantly increased inflammatory activity and no signs of fibrosis in a case with no detectable hepacivirus RNA. Due to highest tissue quality, a terminal hepatic venule instead of a portal triad is shown. D. Recognition of rodent hepacivirus clade 1 antigens by M. glareolus serum. VeroFM cells expressing complete NS3 from <i>M. glareolus</i> hepacivirus RMU10-3382 (GenBank, KC411777) were incubated with 1∶2000-diluted rabbit-anti-NS3 3382 antiserum (control) or 1∶50-diluted rodent serum (picture shows exemplary results for animal NLR 3/C12), followed by goat-anti-rabbit-Cy2 (green) and goat-anti-mouse-Cy3 (red) secondary immunoglobulins. For co-localization analysis of fluorescence signals, the 6th of 12 1-µM Z-stags is shown for every channel. Cross-reactivity with HCV antigens was analyzed by incubation of HuH7 cells, transfected with HCV replicon JFH1, with a rabbit-anti-human-HCV-NS3-49 serum, diluted 1∶400 (control) or rodent serum NLR 3/C12 diluted 1∶50, followed by goat-anti-rabbit-Cy2 (green) and goat-anti-mouse-Cy3 (red) secondary antibodies. Counterstaining was performed using DAPI. Bar, 25 µm.</p

Genomic characterization of the novel rodent hepaciviruses.

<p>A. Partial NS3 gene phylogeny. The analysis comprised a 978 nucleotide fragment of the HCV NS3 gene corresponding to positions 3,912–4,889 in HCV 1a H77 (GenBank, NC_004102). GenBank accession numbers of reference hepaciviruses are indicated to the right of taxon names. Tree topology was inferred using BEAST with a GTR nucleotide substitution model as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#s2" target="_blank">methods section</a>. Rodent hepaciviruses from this study are shown in red and boldface, equine hepaciviruses from this study are shown in blue and boldface. Red squares indicate those viruses whose near full-length genomes were generated. Statistical support of grouping is shown as posterior probabilities at deep nodes. Scale bar corresponds to genetic distance. To the right, 5,000 tree replicates of the same analysis are rendered using Densitree (initial 5,000 trees discarded as burn-in). Green line color indicates low probability of all trees, line thickness corresponds to concordant topologies across tree replicates. B. Genome organization of the novel rodent hepaciviruses. Genes were annotated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#s2" target="_blank">methods section</a>. Black arrows on the top indicate predicted signal peptidase cleavage sites. Red arrows below indicate N-, blue arrows O-glycosylation sites. Putative gene starts and ends are numbered below polyprotein plots. HCV 1a strain H77 is depicted on top as a reference. RMU10-3382 (KC411777) also represents the highly similar virus NLR-365 (KC411796) in <i>M. glareolus</i> hepacivirus clade 1. SAR-46 (KC411807) also represents SAR-3 (KC411806), both from the <i>R. pumilio</i> hepacivirus clade. GenBank accession number of NLR-AP70 representing <i>M. glareolus</i> hepacivirus clade 2 is KC411784. The structural genome region included <i>Core</i> (C), <i>Envelope</i> 1 and 2 (E1 and E2) and p7 genes. The boxes in the HCV and SAR-46 <i>Core</i> gene indicate a putative F protein open reading frame. The putative <i>Core</i> gene at the N′-terminus of the rodent hepacivirus polyproteins included a high number of strongly basic lysine and arginine residues (<i>M. glareolus</i> clade 1, 29 of 163 residues (17.8%); <i>M. glareolus</i> clade 2, 23 of 149 (15.4%); <i>R. pumilio</i> clade, 28 of 172 (16.3%); compared to 31 of 191 (16.2%) in HCV-1a and 11 of the N′-terminal 200 residues (5.5%) in the pegivirus GBV-C1 not encoding a Core protein. Non-structural genes included an NS3 <i>protease</i>/<i>helicase</i> gene, the phosphoprotein NS5a and the NS5b gene encoding the <i>RNA-dependent RNA polymerase</i>. Within the NS4 gene, only the NS4b portion could be clearly identified for all viruses. An NS4a homologue could only be detected in virus SAR-46. IRES types and structural elements in the 3′-genome ends are depicted adjacent to the polyprotein plots for viruses NLR-3382 and SAR-46. Of NLR-AP70, only the 5′-end could be partially determined. This sequence was almost identical to RMU10-3382/NLR-365. The 3′-terminus of NLR-AP70 could not be determined.</p

Complete polyprotein gene phylogeny and comparison of the genome termini between the novel rodent and prototype hepaciviruses.

<p>For the Bayesian phylogeny shown to the left, the WAG amino acid substitution model was used in MrBayes as indicated in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#s2" target="_blank">methods section</a>. Statistical support of grouping from Bayesian posterior probabilities is indicated at node points. Scale bar corresponds to genetic distance. The Pestivirus BVDV (NC_001461) was chosen as an outgroup and truncated for graphical reasons. Branches leading to the novel hepaciviruses from this study are in orange. GenBank accession numbers of analyzed hepaciviruses correspond to those indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat-1003438-g003" target="_blank"><b>Figure 3A</b></a>. The 5′- and 3′-genome termini were re-drawn from published foldings for equine hepaciviruses <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat.1003438-Burbelo1" target="_blank">[24]</a>, HCV <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat.1003438-Thurner1" target="_blank">[63]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat.1003438-Friebe1" target="_blank">[77]</a> and GBV-B <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat.1003438-Rijnbrand1" target="_blank">[78]</a> and <i>de novo</i> for this study for the 5′- and 3′-ends of GBV-C1 and the 3′-end of GBV-B (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat.1003438.s004" target="_blank">Supplementary Figure 4</a></b>). Despite earlier attempts to fold the 3′-ncr of GBV-B <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003438#ppat.1003438-Bukh3" target="_blank">[21]</a> only the 3′-terminal stem-loop structure of GBV-B could be reliably folded due to the single sequence available. The folding of the 3′-end of RMU10-3382 remained tentative for the same reason. No sequence information was available for the 3′-ends of the canine/equine hepacivirus clade and <i>M. glareolus</i> hepacivirus clade 2 (indicated as “n.a.”). Typical pegivirus domains are highlighted in blue and ordered by arabian numbers. Typical hepacivirus domains are highlighted in orange and numbered by roman numbers.</p