Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage

© 2012 Millet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes

Dyspnea assessment and adverse events during sputum induction in COPD

BACKGROUND: The inhalation of normal or hypertonic saline during sputum induction (SI) may act as an indirect bronchoconstrictive stimulus leading to dyspnea and lung function deterioration. Our aim was to assess dyspnea and adverse events in COPD patients who undergo SI following a safety protocol. METHODS: Sputum was induced by normal and hypertonic (4.5%) saline solution in 65 patients with COPD of varying severity. In order to minimize saline-induced bronchoconstriction a protocol based on the European Respiratory Society sputum induction Task group report was followed. Dyspnea change was scored using the Borg scale and lung function was assessed by spirometry and oximetry. RESULTS: Borg score changes [median(IQR) 1.5(0–2)] were observed during SI in 40 subjects; 16 patients required temporary discontinuation of the procedure due to dyspnea-general discomfort and 2 did not complete the session due to dyspnea-wheezing. The change in Borg dyspnea score was significantly correlated with oxygen saturation and heart rate changes and with discontinuation of the procedure due to undesired symptoms. 19 subjects presented an hyperresponsive reaction (decline>20% from baseline FEV(1)). No significant correlation between Borg changes and FEV(1)decline was found. Patients with advanced COPD presented significantly greater Borg and oxygen saturation changes than patients with less severe disease (p = 0.02 and p = 0.001, respectively). Baseline FEV(1), oxygen saturation and 6MWT demonstrated significant diagnostic values in distinguishing subjects who develop an adverse physiologic reaction during the procedure. CONCLUSION: COPD patients undergoing SI following a safety protocol do not experience major adverse events. Dyspnea and oxygen desaturation is more likely to occur in patients with disease in advanced stages, leading to short discontinuation or less frequently to termination of the procedure. Baseline FEV(1), oxygen saturation and 6MWT may have a prognostic value for the development of these adverse events and might be useful to be evaluated in advance

Identification of miRNA-103 in the Cellular Fraction of Human Peripheral Blood as a Potential Biomarker for Malignant Mesothelioma – A Pilot Study

Background: To date, no biomarkers with reasonable sensitivity and specificity for the early detection of malignant mesothelioma have been described. The use of microRNAs (miRNAs) as minimally-invasive biomarkers has opened new opportunities for the diagnosis of cancer, primarily because they exhibit tumor-specific expression profiles and have been commonly observed in blood of both cancer patients and healthy controls. The aim of this pilot study was to identify miRNAs in the cellular fraction of human peripheral blood as potential novel biomarkers for the detection of malignant mesothelioma. Methodology/Principal Findings: Using oligonucleotide microarrays for biomarker identification the miRNA levels in the cellular fraction of human peripheral blood of mesothelioma patients and asbestos-exposed controls were analyzed. Using a threefold expression change in combination with a significance level of p,0.05, miR-103 was identified as a potential biomarker for malignant mesothelioma. Quantitative real-time PCR (qRT-PCR) was used for validation of miR-103 in 23 malignant mesothelioma patients, 17 asbestos-exposed controls, and 25 controls from the general population. For discrimination of mesothelioma patients from asbestos-exposed controls a sensitivity of 83 % and a specificity of 71 % were calculated, and for discrimination of mesothelioma patients from the general population a sensitivity of 78 % and a specificity of 76%

Evolution of the TGF-β Signaling Pathway and Its Potential Role in the Ctenophore, Mnemiopsis leidyi

The TGF-β signaling pathway is a metazoan-specific intercellular signaling pathway known to be important in many developmental and cellular processes in a wide variety of animals. We investigated the complexity and possible functions of this pathway in a member of one of the earliest branching metazoan phyla, the ctenophore Mnemiopsis leidyi. A search of the recently sequenced Mnemiopsis genome revealed an inventory of genes encoding ligands and the rest of the components of the TGF-β superfamily signaling pathway. The Mnemiopsis genome contains nine TGF-β ligands, two TGF-β-like family members, two BMP-like family members, and five gene products that were unable to be classified with certainty. We also identified four TGF-β receptors: three Type I and a single Type II receptor. There are five genes encoding Smad proteins (Smad2, Smad4, Smad6, and two Smad1s). While we have identified many of the other components of this pathway, including Tolloid, SMURF, and Nomo, notably absent are SARA and all of the known antagonists belonging to the Chordin, Follistatin, Noggin, and CAN families. This pathway likely evolved early in metazoan evolution as nearly all components of this pathway have yet to be identified in any non-metazoan. The complement of TGF-β signaling pathway components of ctenophores is more similar to that of the sponge, Amphimedon, than to cnidarians, Trichoplax, or bilaterians. The mRNA expression patterns of key genes revealed by in situ hybridization suggests that TGF-β signaling is not involved in ctenophore early axis specification. Four ligands are expressed during gastrulation in ectodermal micromeres along all three body axes, suggesting a role in transducing earlier maternal signals. Later expression patterns and experiments with the TGF-β inhibitor SB432542 suggest roles in pharyngeal morphogenesis and comb row organization