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

The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells

HIV fusion and entry into CD4 T cells are mediated by two receptors, CD4 and CXCR4. This receptor requirement can be abrogated by pseudotyping the virion with the vesicular stomatitis virus glycoprotein (VSV-G) that mediates viral entry through endocytosis. The VSV-G-pseudotyped HIV is highly infectious for transformed cells, although the virus circumvents the viral receptors and the actin cortex. In HIV infection, gp120 binding to the receptors also transduces signals. Recently, we demonstrated a unique requirement for CXCR4 signaling in HIV latent infection of blood resting CD4 T cells. Thus, we performed parallel studies in which the VSV-G-pseudotyped HIV was used to infect both transformed and resting T cells in the absence of coreceptor signaling. Our results indicate that in transformed T cells, the VSV-G-pseudotyping results in lower viral DNA synthesis but a higher rate of nuclear migration. However, in resting CD4 T cells, only the HIV envelope-mediated entry, but not the VSV-G-mediated endocytosis, can lead to viral DNA synthesis and nuclear migration. The viral particles entering through the endocytotic pathway were destroyed within 1–2 days. These results indicate that the VSV-G-mediated endocytotic pathway, although active in transformed cells, is defective and is not a pathway that can establish HIV latent infection of primary resting T cells. Our results highlight the importance of the genuine HIV envelope and its signaling capacity in the latent infection of blood resting T cells. These results also call for caution on the endocytotic entry model of HIV-1, and on data interpretation where the VSV-G-pseudotyped HIV was used for identifying HIV restriction factors in resting T cells

Quantitative Phosphoproteomics of CXCL12 (SDF-1) Signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation

The role of cytoskeleton and membrane dynamics in HIV-1 entry and infection

Background: In HIV-1 viral fusion and infection, F-actin is required for CD4-co-receptor redistribution in viral cell contact areas, but little is known about the molecular mechanisms un- derlying this fundamental process. We investigated whether HIV-1 Env might promote viral entry by activating ERM (ezrin- radixin-moesin) proteins in a PI4P5-K Ialpha-mediated PIP2 production-dependent manner to regulate F-actin reorganization and CD4/co-receptors clustering and direct interaction. Observations: In this study, we show that HIV-1 virus promotes pore fusion formation and viral infection by inducing PIP2 pro- duction. This process appeared to be mediated by the PI4P5-K Ia kinase. Over-expression of wtPI4P5-K Ia increased HIV-1 Env-mediated PIP2 production and enhanced viral fusion and replication, in permissive lymphocytes. PIP2 production and HIV-1 infection were reduced in cells over-expressing the kinase-dead mutant D227A (D/A)-PI4P5-K Ia, or after knock- down of endogenous PI4P5-K Ia. Moreover, X4-tropic HIV-1 viral fusion and infection required the activation of moesin, an actin adaptor protein of the ERM family, in a PIP2-dependent manner. HIV-1-gp120-induced CD4/CXCR4 association and clustering, occurred during early viral entry, and required moesin-mediat- ed plasma membrane-actin anchoring. Suppression of moesin impeded HIV-1-envelope-mediated F-actin reorganization, CD4/ CXCR4 clustering and interaction, and inhibits HIV-1 entry and infection in lymphocytes. Remarkably, moesin-specific silencing or a dominant-negative construct alters the trafficking of nascent endocytic clatrhin-coated vesicles, which accumulates near the plasma membrane carrying the TfR receptor. Conclusions: · PI4P5-K Ialpha-mediated PIP2 production is involved in the regulation of HIV-1 viral infection. · Moesin is involved in the control of trafficking of CCVs and activated moesin promotes F-actin redistribution and CD4/CXCR4 clus- tering is required for X4-tropic HIV-1 infection in permissive lymphocytes

Modelo inductivo de enseñanza-aprendizaje

Comprobar la eficacia de unidades didácticas elaboradas siguiendo un método inductivo de acuerdo al modelo propuesto por H. Taba (1963). Examinar la relación entre las conductas inductivas emitidas por el profesor y el rendimiento del alumnado. Ofrecer una alternativa de diseño de unidades didácticas, sensibilizando a los profesores sobre el ritmo de aprendizaje peculiar de cada alumno. Participan 15 profesores de EGB y 497 alumnos de los 8 niveles de EGB del Colegio Nacional Centro Piloto Juan Ramón Jiménez, e ICE de la Universidad de Sevilla. En la evaluación de actitudes a través de un cuestionario se utilizan 150 ss. elegidos al azar de la segunda etapa de EGB. Diseño pretest posttest de un sólo grupo. Se controlaron algunas variables extrañas, pudiendose incluir en el tipo cuasirrepresentativo en la taxonomía de Snow (1974). Variable independiente: estrategia inductiva operativizada en unidades didácticas, y los comportamientos docentes del profesor. Variable dependiente: rendimiento del alumno. Fases del trabajo: entrevista con los profesores, tras la cual cada uno elabora su unidad didáctica de acuerdo a un modelo común y las pruebas de rendimiento objetivo que se aplican antes y después de la unidad didáctica. En la última fase se grabaron en video 30 minutos de clase. La información se categorizó según 3 instrumentos de observación. Se diseñaron gráficas 'time line', basadas en los sistemas de Flanders (1977), que reflejan las secuencias de las categorías observacionales recogidas en los 3 instrumentos. Concluye con los datos de un cuestionario que evalúa las actitudes de indagación adquiridas por el alumno durante la experiencia. Diferencias significativas entre el pretest y el posttest en casi todas las clases experimentales. Se constató que el rendimiento de algunos profesores era superior al resto. De las categorías de observación se extrajo que en el instrumento Taba, la categoría de generalización era la más adecuada para predecir el rendimiento. La categoría explicación inferencial correlacionaba negativamente. Con el CLAIM la categoría procesamieto de datos no es relevante. En el Aschner-Gallager solo la categoría de memoria tuvo una correlacion elevada. Del cuestionario se deduce que hay una mayor aceptación, por parte del alumnado, del método de indagación, mayor en octavo y menor en sexto. El modelo de Taba no provocó un rendimiento significativo, el comportamiento inductivo del profesor no correlacionó significativamente con el rendimiento de los alumnos. Sugerencias sobre la utilidad del modelo de Taba en la programación didáctica, y recomendaciones metolódogicas sobre recogida de datos, control experimental, registro de observaciones, definición de variables, diseño y necesidad de entrenamiento en conductas inductivas por parte del profesor.AndalucíaBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín, 5 - 3 Planta; 28014 Madrid; Tel. +34917748000; Fax +34917748026; [email protected]