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

An international randomised controlled trial to compare TARGeted Intraoperative radioTherapy (TARGIT) with conventional postoperative radiotherapy after breast-conserving surgery for women with early-stage breast cancer (the TARGIT-A trial)

Background: Based on our laboratory work and clinical trials we hypothesised that radiotherapy after lumpectomy for breast cancer could be restricted to the tumour bed. In collaboration with the industry we developed a new radiotherapy device and a new surgical operation for delivering single-dose radiation to the tumour bed – the tissues at highest risk of local recurrence. We named it TARGeted Intraoperative radioTherapy (TARGIT). From 1998 we confirmed its feasibility and safety in pilot studies. Objective: To compare TARGIT within a risk-adapted approach with whole-breast external beam radiotherapy (EBRT) over several weeks. Design: The TARGeted Intraoperative radioTherapy Alone (TARGIT-A) trial was a pragmatic, prospective, international, multicentre, non-inferiority, non-blinded, randomised (1 : 1 ratio) clinical trial. Originally, randomisation occurred before initial lumpectomy (prepathology) and, if allocated TARGIT, the patient received it during the lumpectomy. Subsequently, the postpathology stratum was added in which randomisation occurred after initial lumpectomy, allowing potentially easier logistics and a more stringent case selection, but which needed a reoperation to reopen the wound to give TARGIT as a delayed procedure. The risk-adapted approach meant that, in the experimental arm, if pre-specified unsuspected adverse factors were found postoperatively after receiving TARGIT, EBRT was recommended. Pragmatically, this reflected how TARGIT would be practised in the real world. Setting: Thirty-three centres in 11 countries. Participants: Women who were aged ≥ 45 years with unifocal invasive ductal carcinoma preferably ≤ 3.5 cm in size. Interventions: TARGIT within a risk-adapted approach and whole-breast EBRT. Main outcome measures: The primary outcome measure was absolute difference in local recurrence, with a non-inferiority margin of 2.5%. Secondary outcome measures included toxicity and breast cancer-specific and non-breast-cancer mortality. Results: In total, 3451 patients were recruited between March 2000 and June 2012. The following values are 5-year Kaplan–Meier rates for TARGIT compared with EBRT. There was no statistically significant difference in local recurrence between TARGIT and EBRT. TARGIT was non-inferior to EBRT overall [TARGIT 3.3%, 95% confidence interval (CI) 2.1% to 5.1% vs. EBRT 1.3%, 95% CI 0.7% to 2.5%; p = 0.04; Pnon-inferiority = 0.00000012] and in the prepathology stratum (n = 2298) when TARGIT was given concurrently with lumpectomy (TARGIT 2.1%, 95% CI 1.1% to 4.2% vs. EBRT 1.1%, 95% CI 0.5% to 2.5%; p = 0.31; Pnon-inferiority = 0.0000000013). With delayed TARGIT postpathology (n = 1153), the between-group difference was larger than 2.5% and non-inferiority was not established for this stratum (TARGIT 5.4%, 95% CI 3.0% to 9.7% vs. EBRT 1.7%, 95% CI 0.6% to 4.9%; p = 0.069; Pnon-inferiority = 0.06640]. The local recurrence-free survival was 93.9% (95% CI 90.9% to 95.9%) when TARGIT was given with lumpectomy compared with 92.5% (95% CI 89.7% to 94.6%) for EBRT (p = 0.35). In a planned subgroup analysis, progesterone receptor (PgR) status was found to be the only predictor of outcome: hormone-responsive patients (PgR positive) had similar 5-year local recurrence with TARGIT during lumpectomy (1.4%, 95% CI 0.5% to 3.9%) as with EBRT (1.2%, 95% CI 0.5% to 2.9%; p = 0.77). Grade 3 or 4 radiotherapy toxicity was significantly reduced with TARGIT. Overall, breast cancer mortality was much the same between groups (TARGIT 2.6%, 95% CI 1.5% to 4.3% vs. EBRT 1.9%, 95% CI 1.1% to 3.2%; p = 0.56) but there were significantly fewer non-breast-cancer deaths with TARGIT (1.4%, 95% CI 0.8% to 2.5% vs. 3.5%, 95% CI 2.3% to 5.2%; p = 0.0086), attributable to fewer deaths from cardiovascular causes and other cancers, leading to a trend in reduced overall mortality in the TARGIT arm (3.9%, 95% CI 2.7% to 5.8% vs. 5.3%, 95% CI 3.9% to 7.3%; p = 0.099]. Health economic analyses suggest that TARGIT was statistically significantly less costly than EBRT, produced similar quality-adjusted life-years, had a positive incremental net monetary benefit that was borderline statistically significantly different from zero and had a probability of \u3e 90% of being cost-effective. There appears to be little uncertainty in the point estimates, based on deterministic and probabilistic sensitivity analyses. If TARGIT were given instead of EBRT in suitable patients, it might potentially reduce costs to the health-care providers in the UK by £8–9.1 million each year. This does not include environmental, patient and societal costs. Limitations: The number of local recurrences is small but the number of events for local recurrence-free survival is not as small (TARGIT 57 vs. EBRT 59); occurrence of so few events (\u3c 3.5%) also implies that both treatments are effective and any difference is unlikely to be large. Not all 3451 patients were followed up for 5 years; however, more than the number of patients required to answer the main trial question (n = 585) were followed up for \u3e 5 years. Conclusions: For patients with breast cancer (women who are aged ≥ 45 years with hormone sensitive invasive ductal carcinoma that is up to 3.5 cm in size), TARGIT concurrent with lumpectomy within a risk-adapted approach is as effective as, safer than and less expensive than postoperative EBRT. Future work: The analyses will be repeated with longer follow-up. Although this may not change the primary result, the larger number of events may confirm the effect on overall mortality and allow more detailed subgroup analyses. The TARGeted Intraoperative radioTherapy Boost (TARGIT-B) trial is testing whether or not a tumour bed boost given intraoperatively (TARGIT) boost is superior to a tumour bed boost given as part of postoperative EBRT. Trial registration: Current Controlled Trials ISRCTN34086741 and ClinicalTrials.gov NCT00983684. Funding: University College London Hospitals (UCLH)/University College London (UCL) Comprehensive Biomedical Research Centre, UCLH Charities, Ninewells Cancer Campaign, National Health and Medical Research Council and German Federal Ministry of Education and Research (BMBF). From September 2009 this project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 20, No. 73. See the NIHR Journals Library website for further project information

Precise let-7 expression levels balance organ regeneration against tumor suppression

The in vivo roles for even the most intensely studied microRNAs remain poorly defined. Here, analysis of mouse models revealed that let-7, a large and ancient microRNA family, performs tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Modest let-7 overexpression abrogated MYC-driven liver cancer by antagonizing multiple let-7 sensitive oncogenes. However, the same level of overexpression blocked liver regeneration, while let-7 deletion enhanced it, demonstrating that distinct let-7 levels can mediate desirable phenotypes. let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR pathway. We found that chronic high-dose let-7 overexpression caused liver damage and degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in tissue repair and tumorigenesis rationalize the tight regulation of this microRNA in development, and have important implications for let-7 based therapeutics. DOI: http://dx.doi.org/10.7554/eLife.09431.00

Regulation of α-Fetoprotein Expression by Nkx2.8

The α-fetoprotein (AFP) gene is an important model of developmental gene silencing and neoplastic gene reactivation. Nkx2.8 is a divergent homeodomain factor originally cloned through its binding to the promoter-coupling element (PCE), a regulatory region upstream of the AFP promoter that mediates stimulation by distant enhancers. Nkx2.8 is the only developmentally regulated factor that has been associated with AFP gene expression. Fetoprotein transcription factor, an orphan nuclear receptor, has also been shown to bind the PCE but is not developmentally regulated. The binding specificities of both families of transcription factor were determined, and overlapping sites for each were defined in the PCE. After modification of nuclear extract and gel shift analysis procedures, Nkx2.8 was identified in six AFP-positive cell lines. Transient-transfection analysis did not show transcriptional stimulation by Nkx2.8 or other active NK2 factors, which only interfered with gene expression. However, two sets of analysis demonstrated the relationship of Nkx2.8 to AFP expression: chromatin immunoprecipitation demonstrated that Nkx2.8 bound to the active AFP promoter, and antisense inhibition of Nkx2.8 mRNA translation selectively reduced expression of both the endogenous human AFP gene and transfected reporters containing the rat AFP promoter