Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1

AbstractHuman milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3′-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6′-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk

Proteomic Analysis of Mitochondrial-Associated ER Membranes (MAM) during RNA Virus Infection Reveals Dynamic Changes in Protein and Organelle Trafficking

<div><p>RIG-I pathway signaling of innate immunity against RNA virus infection is organized between the ER and mitochondria on a subdomain of the ER called the mitochondrial-associated ER membrane (MAM). The RIG-I adaptor protein MAVS transmits downstream signaling of antiviral immunity, with signaling complexes assembling on the MAM in association with mitochondria and peroxisomes. To identify components that regulate MAVS signalosome assembly on the MAM, we characterized the proteome of MAM, ER, and cytosol from cells infected with either chronic (hepatitis C) or acute (Sendai) RNA virus infections, as well as mock-infected cells. Comparative analysis of protein trafficking dynamics during both chronic and acute viral infection reveals differential protein profiles in the MAM during RIG-I pathway activation. We identified proteins and biochemical pathways recruited into and out of the MAM in both chronic and acute RNA viral infections, representing proteins that drive immunity and/or regulate viral replication. In addition, by using this comparative proteomics approach, we identified 3 new MAVS-interacting proteins, RAB1B, VTN, and LONP1, and defined LONP1 as a positive regulator of the RIG-I pathway. Our proteomic analysis also reveals a dynamic cross-talk between subcellular compartments during both acute and chronic RNA virus infection, and demonstrates the importance of the MAM as a central platform that coordinates innate immune signaling to initiate immunity against RNA virus infection.</p></div

Cellular interaction network of proteins identified with dynamic MAM localization during RNA virus replication.

<p>Cellular mapping of the interaction network of proteins identified in MAM fractions that either increase in the MAM with HCV and SenV (yellow), decrease in the MAM with both HCV and SenV (blue), or that have “MAVS-like” localization (red; decreased in the MAM with HCV, increased or unchanged in MAM with SenV). The interaction network was determined by using InnateDb and visualized with Cytoscape. It includes all proteins that interact with at least one other protein. Bold-faced dotted edges represent protein-protein interactions discovered and validated in this study. Previously described MAVS-interacting proteins (MAVS, MFN1, MFN2, C1QBP, AMFR, and TOMM70) are highlighted by large red circles. Nodes representing each protein were positioned on a cell map according to subcellular localization using annotation information from InnateDb.</p

Experimental design for proteomic analysis of MAM fractions during RNA virus replication.

<p>(A) Percoll gradient biochemical fractionation scheme. (B) Electron micrograph of Huh7.5 cells. MAM is indicated by the arrow pointing to the membrane wrapping around a mitochondrion. (C) Percoll gradient illustrating a representative fractionation of PH5CH8 cells at the mitochondria/MAM isolation step. MAM and mitochondria were extracted from the gradient by a needle at the location indicated by the arrow and subjected to further purification. (D) Immunoblot analysis of biochemical fractions isolated from PH5CH8 cells. Fractionation markers: calnexin, ER; Cox-1, mitochondria; FACL4, MAM; tubulin, cytosol. (E) Experimental scheme for proteomic analysis.</p

Gene Ontology Analysis for proteins that leave the MAM during RNA virus.

<p>Shown are the top 5 pathways that are enriched under each condition, the number of proteins that fall within each category (N), and the p-values obtained.</p><p>Gene Ontology Analysis for proteins that leave the MAM during RNA virus.</p

Identification of MAVS-interacting proteins using MAM proteomics.

<p>(A) Venn diagram of proteins that share a MAM-localization profile similar to that of MAVS. Proteins that move out of the MAM fraction during HCV (greater than 2-fold change over mock with a Benjamini-Hochberg corrected p-value of < 0.05) were compared to those that either move into the MAM or remain localized to the MAM during SenV infection. The 281 proteins in the intersection share this MAVS-localization pattern in the MAM during RNA virus infection. (B) Heat map of the 281 proteins with “MAVS-like” localization identified in MAM fractions from HCV and SenV, as well as mock (see panel A). Log₁₀-transformed spectral counts across technical triplicates are represented by the color intensity shown in the key. (C) Immunoblot analysis of lysate (Input) and anti-Myc immunoprecipated extracts (Pellet) from cells expressing Myc-MAVS (+) or vector (-), and either Flag-RAB1B, Flag-VTN, or Flag-LONP1. (D) IFN-β promoter reporter luciferase expression of Huh7 cells expressing empty vector or Flag-LONP1 and then mock or SenV infected (24h). Values are mean -/+ SD (n = 3) of one of three replicate experiments; **P<0.01. (Inset) Immunoblot for Flag-LONP1 and tubulin (Tub.) protein expression.</p

Network enrichment map of protein dynamics on the MAM during RNA virus replication.

<p>Enrichment map for proteins moving into the MAM with SenV (A) or leaving the MAM with SenV (B), compared to the proteins identified on the MAM during HCV. Nodes represent enriched biological pathways grouped by DAVID-identified clusters, manually circled and classified by function, for proteins differentially expressed in the MAM fraction following SenV (≥2-fold; BH-adjusted p-value < 0.05). Nodes were classified by Functional Annotation Clustering of Gene Ontology (GO) biological pathways using the DAVID bioinformatics resource. Edges between nodes represent overlap between two pathways, and edges between functional category clusters represent at least one shared protein. Node internal color is proportional to the number of proteins comprising the canonical pathway (darker shading in the node means greater number of proteins). Node edges in red represent pathways enriched in the MAM in cells replicating HCV and SenV, and node edges in blue represent pathways enriched in groups of proteins leaving the MAM in cells replicating HCV and SenV. Nodes edges in black represent SenV-specific changes. Node identity is depicted by the letter within each node, and the key is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117963#pone.0117963.s006" target="_blank">S4 Table</a>.</p

Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

El objetivo es presentar categorías de la Teoría Histórico-Cultural de la Actividad como caja de herramientas para construir indagaciones e intervenciones vinculadas al trabajo de Psicólogos y otros Agentes en escenarios educativos. En el marco de problemas cruciales de agenda psicoeducativa, como el de las unidades de análisis, se introduce la Teoría de la Actividad en su primera, segunda y tercera generación y “más allá”, como Engestrom caracterizó su desarrollo histórico. Se presentan similaridades y diferencias con conceptualizaciones de autores de perspectivas socio-cultural e histórico–cultural, originadas en el pensamiento de Vygotsky, como Cole, Rogoff, Wertsch, Chaiklin, Daniels y Edwards, tanto en relación a la Psicología del Desarrollo como al giro relacional en la experticia. Se despliega la Teoría como artefacto mediador para construir investigaciones psicoeducativas en curso sobre violencias en escuelas desde la perspectiva de los actores y participación de psicólogos en configuraciones de apoyo entre Educación Especial y Escuela Común. Finalmente, conceptualizaciones y figuras diseñadas por Engestrom y Yamazumi, en la post-generación de la Teoría, permiten analizar procesos de vinculación e intercambio entre Universidad y Escuelas para la inclusión y calidad educativas, en los que participan psicólogos en formación y formadores de la Universidad Nacional de La Plata.The aim is to present categories of Cultural-Historic-Activity-Theory as a tools cage for the building of research and interventions related to Psychologists and other Agents´ work in educational stages. In the frame of central problems of the psycho-educational agenda, as the units of analysis, the Activity Theory is introduced in its first, second and third generation and beyond, as Engestrom depicted its historical development. Similarities and differences are presented between Activity Theory and conceptualizations of different authors of socio-cultural and cultural-historic perspectives, both founded on Vygotsky thought, linked to Development Psychology and to the relational shift in expertice, as Cole, Rogoff, Wertsch, Chaiklin, Daniels and Edwards. The Activity Theory is displayed as a mediating artifact for building current psycho-educational research about violences at school from the perspective of social actors and about psychologists and other agents´ participation in supportive configurations between Special Education and Common Schools. At last, figures and conceptualizations drawn by Engestrom and Yamazumi in the post-generation of the Theory, allow to analyse processes of linking and exchange between University and Schools for educational enhancement and inclusion, in which psychologists in modeling and tutors of La Plata National University are participating.Facultad de Psicologí