Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus)

Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [(3)H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2-GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix

Post-Maastricht Civil Society and Participatory Democracy

The Treaty of Maastricht and even more so the sceptical public response to the further deepening of European integration triggered a new approach to civil society. This contribution explores why the concept of civil society became so prominent in Brussels and why such high expectations were placed in the democratic strengthening of Europe by involving civil society in EU governance and by starting a civil dialogue. The main part of the contribution presents the findings of an empirical investigation demonstrating and explaining how much aspirations differ from reality. At the end of the contribution the democratic potential of civil dialogue is compared with expectations regarding the European Citizens’ Initiative

Structure of the MxA stalk elucidates the assembly of ring-like units of an antiviral module

The dynamin-like MxA GTP ase (Myxovirus resistance protein 1) mediates cellular resistance against a wide range of viruses. MxA is composed of an amino-terminal G domain, a middle domain and a carboxy-terminal GTPase effector domain. We recently determined the structure of the middle domain and GTPase effector domain of MxA constituting an elongated helical stalk, and elucidated the mechanism how the stalk mediates formation of ring-like MxA oligomers. Here, we shortly review our work and discuss the MxA rings as functional units of a cellular module orchestrating and executing the antiviral response