Cryotomography of budding influenza a virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end

Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process

Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ∼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared with iGluSnFR, iGluu has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons

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

Metropolitan ungovernability

Although most of the population already live in metropolitan areas, in the Brazilian legal framework, there is still no appropriate legal ruling for managing these territories. The federal pact resulting from the Federal Constitution of 1988 established that it was at the prerogative of the states to create metropolitan regions, urban agglomerations and micro-regions, but did not attribute sufficient powers to them so they could conduct public functions of common interest (FPICs, in Portuguese), given the municipal autonomy likewise granted by the Constitution. In no other aspect does this legal conflict appear more evident than in territorial organization, since virtually all the instruments for the control of land use are of exclusively municipal competence. The purpose of this article is to discuss the possibilities of smoothing the way to an understanding on the limits of local autonomy in conurbations, thereby subordinating the “local” interest to the common interest, with regard to the regulating urban occupation. Therefore, we start with a brief analysis of the 1973 Law which set up the first metropolitan areas in Brazil, and thereby seek to identify advances and retrograde steps that the Constitution represented in relation to the division of powers between federal entities with respect to land management. Then, we investigate the role that post-1988 federal law reserves to metropolitan bodies in organizing, planning and carrying out FPICs in order to catch sight of windows of opportunity to bring them into force with regard to regulating the use of land. Finally, we discuss if the Statute of the Metropolis, which has recently been approved, provides the legal framework needed to overcome the antagonisms that criss-cross metropolitan governance.

O paradigma das global cities nas estratégias de desenvolvimento local

Este artigo trata das novas estratégias de desenvolvimento econômico para as cidades no contexto da reestruturação produtiva. Ele enfoca o papel atribuído às cidades como centros de gestão dos fluxos de capitais, o qual permite uma articulação entre o local e o global sem a intermediação das esferas regional e nacional. Primeiramente, apresenta as contribuições teóricas no Terceiro Mundo que mais têm influenciado o debate urbano sobre a emergência de uma economia de fluxos, cuja organização em rede impõe as cidades como nós de conexão. Em seguida, analisa a construção paradigmática das global cities, com base na generalização de alguns pressupostos teóricos e de tendências empiricamente observadas. Finalmente, examina as estratégias que estão sendo difundidas por consultores internacionais e relacionadas à vocação inexorável das cidades para o terciário avançado.

Barra da Tijuca em números: um estudo sobre o licenciamento de construções nos anos 2000 e 2001

Among the areas of the municipality that grew the most in recent years is the region covered by the neighborhoods of Barra da Tijuca, Itanhangá, and Joá – UEP 451. Its population quadrupled in two decades, increasing from 28,528 residents in 1980 to 114,901 in 2000, although it accounts for only 2% of the total population of the municipality, according to the preliminary results of the 2000 Demographic Census released by IBGE.Entre las regiones del municipio que más crecieron en los últimos años se encuentra la zona que abarca los barrios de Barra da Tijuca, Itanhangá y Joá – UEP 451. Su población se cuadruplicó en dos décadas, pasando de 28,528 habitantes en 1980 a 114,901 en 2000, aunque representa solo el 2% de la población total del municipio, según los resultados preliminares del Censo Demográfico 2000, publicados por el IBGE.Entre as regiões do Município que mais cresceram nos últimos anos, encontrase a área abrangida pelos bairros da Barra da Tijuca, Itanhangá e Joá – a UEP 451. Sua população quadruplicou em duas décadas, passando de 28.528 moradores, em 1980, para 114.901, em 2000, embora corresponda a apenas 2% da população total do Município, segundo os resultados preliminares do Censo Demográfico 2000, divulgados pelo IBGE

A cidade contra a favela: a nova ameaça ambiental

Este artigo trata da apropriação do discurso da preservação ambiental para a retomada da discussão sobre remoções de favelas no Rio de Janeiro que haviam sido rechaçadas no processo de redemocratização do país. Depois da favela como foco de epidemias e antro de marginais, a mais nova representação social que vem sendo construída apresenta-a como fator de degradação ambiental. Auxiliada pelo saber técnico-científico que demonstra empiricamente os danos ao meio ambiente causados pelas ocupações irregulares, observa-se a constituição de um movimento conservador que busca pressionar os poderes públicos a reprimi-las, sobretudo nas áreas mais valorizadas da cidade. O presente trabalho se propõe a evidenciar a estratégia discursiva dos principais protagonistas deste movimento, a partir da análise de uma campanha promovida, no ano de 2005, por um importante jornal local, intitulada “Ilegal. E daí?”, e que teve como conseqüência uma ação movida pelo Ministério Público Estadual solicitando à Prefeitura a remoção de 13 áreas favelizadas.

A Dinâmica Imobiliária no entorno do Complexo do Alemão

A virtuous circle of local development resulting from integrated public policies took place in one of the most conflicted areas of Rio de Janeiro: the Complexo do Alemão. The combination of investments in urban infrastructure, housing, social facilities and public safety, promoted by the three governmental spheres in the last ten years in this locality and in its immediate surroundings, resulted in an unequivocal expansion of economic activities and the supply of work and income, and in the real estate valuation inside and outside the favela. The maintenance of this positive momentum, however, is now threatened by the deep political and economic crisis that broke out in the country, particularly in our State. The objective of this article is to analyze the behavior of the real estate market in the region, having as a reference frame the Growth Acceleration Program (PAC), started in 2008, with a view to contributing to a reflection about the possible public strategies for the preservation of the gains achieved.Un círculo virtuoso de desarrollo local, resultado de políticas públicas integradas, tuvo lugar en una de las áreas más conflictivas de Río de Janeiro: el Complexo do Alemão. La combinación de inversiones en infraestructura urbana, vivienda, equipamientos sociales y seguridad pública, promovidas por los tres niveles de gobierno en los últimos diez años en esta localidad y su entorno inmediato, resultó en una inequívoca ampliación de las actividades económicas, la oferta de empleo e ingresos, y en la valorización inmobiliaria dentro y fuera de la favela. Sin embargo, el mantenimiento de esta dinámica positiva se ve hoy amenazado por la profunda crisis política y económica que ha surgido en el país, particularmente en nuestro Estado. El objetivo del presente artículo es analizar el comportamiento del mercado inmobiliario en la región, tomando como marco de referencia el Programa de Aceleración del Crecimiento (PAC), iniciado en 2008, con el fin de contribuir a una reflexión sobre posibles estrategias públicas para preservar los logros sociales alcanzados.Um círculo virtuoso de desenvolvimento local decorrente de políticas públicas integradas teve lugar em uma das áreas mais conflagradas do Rio de Janeiro: o Complexo do Alemão. A combinação de investimentos em infraestrutura urbana, habitação, equipamentos sociais e segurança pública, promovidos pelas três esferas governamentais nos últimos dez anos nesta localidade e no seu entorno imediato, resultou em inequívoca ampliação das atividades econômicas e da oferta de trabalho e renda, e na valorização imobiliária dentro e fora da favela. A manutenção desta dinâmica positiva, contudo, é hoje ameaçada pela profunda crise política e econômica irrompida no país, em particular em nosso Estado. O objetivo do presente artigo é analisar o comportamento do mercado imobiliário na região, tendo como marco referencial o Programa de Aceleração do Crescimento (PAC), iniciado em 2008, com vistas a contribuir para uma reflexão acerca das possíveis estratégias públicas para a preservação dos ganhos sociais conquistados

Biochemical and Structural Evidence in Support of a Coherent Model for the Formation of the Double-Helical Influenza A Virus Ribonucleoprotein

Influenza A virions contain eight ribonucleoproteins (RNPs), each comprised of a negative-strand viral RNA, the viral polymerase, and multiple nucleoproteins (NPs) that coat the viral RNA. NP oligomerization along the viral RNA is mediated largely by a 28-amino-acid tail loop. Influenza viral RNPs, which serve as the templates for viral RNA synthesis in the nuclei of infected cells, are not linear but rather are organized in hairpin-like double-helical structures. Here we present results that strongly support a coherent model for the assembly of the double-helical influenza virus RNP structure. First, we show that NP self-associates much more weakly in the absence of RNA than in its presence, indicating that oligomerization is very limited in the cytoplasm. We also show that once NP has oligomerized, it can dissociate in the absence of bound RNA, but only at a very slow rate, indicating that the NP scaffold remains intact when viral RNA dissociates from NPs to interact with the polymerase during viral RNA synthesis. In addition, we identify a previously unknown NP-NP interface that is likely responsible for organizing the double-helical viral RNP structure. This identification stemmed from our observation that NP lacking the oligomerization tail loop forms monomers and dimers. We determined the crystal structure of this NP dimer, which reveals this new NP-NP interface. Mutation of residues that disrupt this dimer interface does not affect oligomerization of NPs containing the tail loop but does inactivate the ability of NPs containing the tail loop to support viral RNA synthesis in minigenome assays