Los autores desean agradecer a Nada Naffakh y Marie-Anne Rameix-Welti (Instituto Pasteur) para el virus RWSN-PB2-Strep.

<html> Un diagrama de flujo esquemático del protocolo se muestra en la fig. 1 y una tabla de los reactivos se presentan a continuación. 1. Infección (16 - 24 h) <ol><li> Semilla a las células HeLa en 5 150 mm platos de plástico de cultivo de células en un medio de Dulbecco modificado de Eagle (DMEM)-medio-alto de glucosa, de tal manera que alcanzará aproximadamente el 80% de confluencia al día siguiente (aproximadamente 5 x 10 8 células). Es importante que las células no llegan a 100% de confluencia. </li><li> Al día siguiente, se diluye la Strep-etiquetados stock de virus de la gripe en salina tamponada con fosfato (PBS) que contenía 0,3% de albúmina sérica bovina (BSA) a una multiplicidad de infección de 3. </li><li> Eliminar el medio de crecimiento de las células, se lava una vez con PBS, y se añaden 10 ml de PBS que contenía el virus a las células. Incubar durante 1 hora a temperatura ambiente. </li><li> Sustituir el medio de infección con DMEM-alto de glucosa, y continuar la incubación a 37 ° C. </li></ol>&quot;&gt; La duración de la incubación depende de la fase de la infección a estudiar (ver Discusión). 2. Fraccionamiento subcelular (3 h) <ol><li> Lavar las células una vez con PBS frío y luego suspender las células en PBS frío con una espátula de goma y la transferencia a un tubo cónico de 50 ml. </li><li> Las células de pellets en una centrífuga de mesa durante 5 min a 1.000 rpm y luego aspirar el PBS. </li><li> Resuspender pellet de células en 10 ml de tampón de sacarosa frío (10 mM, pH 7,9 HEPES, 10 mM de KCl, 2 mM de acetato de Mg (MgOAc), 3 mM de CaCl 2, 340 mM de sacarosa, 1 mM de ditiotreitol (DTT), 1% de mezcla de inhibidor de la proteasa) . Después de la suspensión, pasar las células a través de una punta de pipeta 200 l unido a un plástico de 10 ml de suero pipeta para interrumpir grupos de células. </li><li> Incubar 10 minutos en hielo. Invierta suavemente periódicamente para volver a suspender las células. </li><li> Añadir Nonidet P-40 (NP-40) a una concentración final de 0,5% y vórtice a alta velocidad durante 15 segundos. Inmediatamente centrifugar durante 10 minutos a 3500 xg a 4 ° C en un centrífugas de mesage. </li><li> Eliminar el sobrenadante y se almacena a 4 º C. Esta es la fracción citoplásmica. El sedimento debe ser más pequeño y más blanca que la célula original de pellets. Todas las fracciones se pueden almacenar por lo menos durante 4 horas a 4 ° C. </li><li> Lavar el precipitado en 5 ml de tampón frío sacarosa, de re-centrífuga como en el paso 2,5 y descartar el sobrenadante. </li><li> Resuspender el sedimento (que contiene núcleos) en 1,5 ml de tampón de extracción en frío nucleoplasmic (HEPES 50 mM, pH 7,9, 150 mM de KOAc, 1,5 mM MgCl 2, 0,1% de NP-40, 1 mM de DTT, 1% de mezcla de inhibidor de la proteasa). </li><li> Pasar a un frío, en cristal, 4 ml Dounce homogeneizadora con una mano apretada y homogeneizar los núcleos con 20 golpes. Evite la formación de espuma durante la homogeneización. La resistencia debe aumentar después de aproximadamente 10 golpes. Homogeneización eficiente puede ser confirmado con un microscopio de contraste de fase. </li><li> Transferir los núcleos homogeneizada a un tubo de microcentrífuga de 1,5 ml y se incuba 20 minutos a una rueda que gira a 4 °C. </li><li> Centrifugar a 16.000 x g en una microcentrífuga a 4 ° C durante 10 min. </li><li> Eliminar el sobrenadante y se almacena a 4 º C. Esta es la fracción nucleoplasmic. </li><li> Resuspender el precipitado en 1,5 ml de tampón de digestión (50 mM HEPES pH 7,9, 10 mM de NaCl, 1,5 mM MgCl2, 1 mM DTT, 1% de mezcla inhibidor de la proteasa, 100 U / ml (Benzonase para el análisis de ARN, sustituto con 20 U / ml de RNasa libre de DNasa I)) precalentado a 37 ° C, y se incuba 10 minutos a 37 ° C. </li><li> Añadir 42 l de NaCl 5 M (para la concentración final de 150 mM de NaCl) y se incuba 20 minutos más a 4 ° C. </li><li> Centrifugar a 16.000 x g en una microcentrífuga a 4 ° C durante 10 min. </li><li> Eliminar el sobrenadante y se almacena a 4 º C. Esta es la fracción CH150. </li><li> Resuspender el pellet en 1,5 ml de frío tampón de alta salinidad (50 mM, pH 7,9 HEPES, 500 mM de NaCl, 1,5 mM de MgCl 2, 0,1% de NP-40, 1 mM de DTT, 1% de mezcla de inhibidor de proteasa) y se incuba 20 minutos en hielo. </li><li> Centrifugar a 16.000 x g &lt;/ Em&gt; en una microcentrífuga a 4 ° C durante 10 min. </li><li> Eliminar el sobrenadante y se almacena a 4 º C. Esta es la fracción ch500. </li></ol> 3. Strep-etiqueta Purificación (2 h) <ol><li> Añadir 300 l de NaCl 5 M a la fracción citoplásmica (para la concentración final de 150 mM de NaCl). </li><li> Alícuota de 100 l de concentrado de Strep-Tactin Sefarosa por fracción en un tubo cónico de 15 ml. </li><li> Añadir 10 volúmenes de tampón de lavado Strep (20 mM, pH 7,9 HEPES, 150 mM de NaCl (500 mM de NaCl para ch500 muestras), 1 mM EDTA) a perlas, invertir el tubo para mezclar, y centrifugar 5 min a 1.000 x g en una mesa centrifugar. </li><li> Descartar el sobrenadante, repita el paso 3,3 dos veces y luego volver a suspender el pellet de talón en un volumen igual de tampón de lavado Strep. </li><li> Añadir 200 l de lavado Strep-Tactin suspensión cordón a cada fracción (en tubos de 1,5 ml para microcentrífuga nuceloplasmic y CH150, y ch500 fracciones y un tubo cónico de 15 ml para la fracción citoplasmática). </li><li> Girartubos de 1 hora a 4 ° C en una rueda giratoria. </li><li> Centrifugar los tubos de 1 min a 1000 x g a 4 ° C. Desechar el sobrenadante. </li><li> Añadir 1 ml de solución de lavado en frío por estreptococo a cada tubo. Transferencia de cuentas de tubo de 15 ml (fracción citoplasmática) al tubo de 1,5 ml. Lavar todos los tubos de 1 min a 4 ° C, después de centrifugación como en el paso 3,7. Repita este paso de lavado dos veces. </li><li> Después del último lavado, eliminar todos los rastros de tampón de lavado utilizando una pipeta de punta plana. Añadir 100 l de buffer de elución (tampón de lavado que contiene Strep 2,5 mM destiobiotina) y mezclar suavemente. </li><li> Incubar 15 minutos en hielo. Mezclar suavemente perlas de vez en cuando tocando el fondo de los tubos. </li><li> Tubos de centrífuga 1 min a 1000 x g a 4 ° C en microcentrífuga. Recoger el líquido sobrenadante que contienen eluidas Strep-etiquetados vRNPs. </li></ol> 4. Los resultados representativos La eficacia del fraccionamiento se juzga mejor por análisis de transferencia Western de los lisados ​​utilizando fraccionadas ANTIBodies específicos para los marcadores subcelulares (Fig. 2). Específicamente, el fraccionamiento subnuclear éxito debería muestran poca o ninguna celular ARN polimerasa II (Pol II) en la nucleoplasmic, o fracción soluble nuclear 4, y la mayoría se extrajo con 150 mM de NaCl 5. Una degradación de Pol II es también reproducible observada en las células infectadas por virus de la gripe en comparación con las células no infectadas, en consonancia con la literatura 6. Purificación de vRNPs se juzga mejor por la plata o tinción de Coomassie, como se muestra para un eluato citoplásmica en la figura. 3. En nuestra experiencia, la mayoría Strep-PB2 se purifica como parte de un vRNP totalmente formado, es decir, soluble polimerasa poco es capturado. Esto se refleja en el PN de alta: PA/PB1/PB2 relación observada por la plata o la tinción de Coomassie. Una proporción menor sugiere contaminación tampón con RNasas, como varios ARNasas ubicuas (como RNasa A) se sabe que el ARN viral escinden de tal manera que la disociaciónse comió la polimerasa de la NP multímeros 7. Curiosamente, el tratamiento con Benzonase solo, mientras que suficiente para digerir ARN viral, no parece tener un efecto en las proporciones estequiométricas de proteínas vRNP. A pesar de que no se puede explicar este fenómeno, se observó alteración de vRNPs después de la digestión con RNasas otros (datos no mostrados), sugiriendo que Benzonase puede dejar algunas regiones importantes de la estructura del ARN intacto. Después de la purificación de vRNPs desde el citoplasma y nucleoplasma (realizado sin digestión con nucleasa), 8 bandas débiles pero discreto en altos pesos moleculares pueden ser observadas por tinción con plata, que corresponden a los pesos moleculares predichos de los 8 segmentos del genoma de la gripe (ver ref. 3 ). Las cantidades relativas de vRNPs purificadas a partir de cada fracción a las 9 horas después de la infección se muestran en la figura. 4. La distribución de vRNPs varía durante el curso de la infección, con fuerte acumulación en la fracción CH150 en puntos de tiempo primeros, yuna mayor acumulación en el nucleoplasma y el citoplasma en momentos finales. <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo del fraccionamiento subcelular. Adaptado de ref. 3. <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="Figura 2"/> Figura 2. Análisis Western blot de fracciones subcelulares de la influenza células infectadas por virus. 10 9 células HeLa fueron infectados con la gripe WSN cepa del virus durante 9 horas antes del fraccionamiento subcelular. Igual cantidad de proteína total fueron cargados en cada carril y se analizaron con los anticuerpos que aparecen. ARN polimerasa II (Pol II) se detectó utilizando clon 8WG16, que reconoce todas las formas del dominio C-terminal (la banda hipofosforilada es más prominente). Adaptado de ref. 3. <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="Figura 3"/> Figura 3.Análisis de tinción de plata de la vRNPs purificada. Se infectaron células HeLa con RWSN (como un control negativo) o RWSN-PB2-Strep durante 9 h, seguido de purificación por estreptococo de la fracción citoplásmica. Asterisco indica que las bandas que no son visibles después de la digestión con RNasa. Adaptado de ref. 3. <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="Figura 4"/> Figura 4. Análisis de tinción de plata de la vRNPs purificados a partir de diferentes fracciones celulares. 10 9 células HeLa fueron infectados con RWSN-PB2-estreptococo o RWSN durante 9 horas seguidas por fraccionamiento subcelular y purificación Strep. Cantidades iguales de eluato de cada fracción se cargaron. Adaptado de ref. 3.

<ol>
	<li>Engelhardt, O. G., Smith, M., Fodor, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+influenza+A+virus+RNA-dependent+RNA+polymerase+with+cellular+RNA+polymerase+II.">Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II.</a> J. Virol. 79, 5812-5812 (2005).</li><li>Garcia-Robles, I., Akarsu, H., Müller, C. W., Ruigrok, R., Baudin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+influenza+virus+proteins+with+nucleosomes.">Interaction of influenza virus proteins with nucleosomes.</a> Virology. 332, 329 (2005).</li><li>Chase, G. P., Rameix-Welti, M. A., Zvirbilene, A., Zvirblis, G., Götz, V., Wolff, T., Naffakh, N., Schwemmle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+ribonucleoprotein+complexes+gain+preferential+access+to+cellular+export+machinery+through+chromatin+targeting.">Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting.</a> PLoS Pathogens. 7, e1002187 (2011).</li><li>Kimura, H., Tao, Y., Roeder, R. G., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+RNA+polymerase+II+and+its+transcription+factors+in+an+HeLa+cell:+little+soluble+holoenzyme+but+significant+amounts+of+polymerases+attached+to+the+nuclear+substructure.">Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure.</a> Mol. Cell. Biol. 19, 5383-5383 (1999).</li><li>Henikoff, S., Henikoff, J. G., Sakai, A., Loeb, G. B., Ahmad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiling+of+salt+fractions+maps+physical+properties+of+chromatin.">Genome-wide profiling of salt fractions maps physical properties of chromatin.</a> Genome Res. 19, 460 (2009).</li><li>Rodriguez, A., Perez-Gonzalez, A., Nieto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+infection+causes+specific+degradation+of+the+largest+subunit+of+cellular+RNA+polymerase+II.">Influenza virus infection causes specific degradation of the largest subunit of cellular RNA polymerase II.</a> J. Virol. 81, 5315-5315 (2007).</li><li>Ye, Z., Liu, T., Offringa, D. P., McInnis, J., Levandowski, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+influenza+virus+matrix+protein+with+ribonucleoproteins.">Association of influenza virus matrix protein with ribonucleoproteins.</a> J. Virol. 73, 7467-7467 (1999).</li><li>Watanabe, T., Watanabe, S., Kawaoka, T., Y, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+networks+involved+in+the+influenza+virus+life+cycle.">Cellular networks involved in the influenza virus life cycle.</a> Cell Host Microbe. 7, 427 (2010).</li><li>Engelhardt, O. G., Fodor, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+association+between+viral+and+cellular+transcription+during+influenza+virus+infection.">Functional association between viral and cellular transcription during influenza virus infection.</a> Rev. Med. Virol. 16, 329-345 (2006).</li><li>Schwartz, L. B., Sklar, V. E., Jaehning, J. A., Weinmann, R., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+partial+characterization+of+the+multiple+forms+of+deoxyribonucleic+acid-dependent+ribonucleic+acid+polymerase+in+the+mouse+myeloma,+MOPC+315.">Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the mouse myeloma, MOPC 315.</a> J. Biol. Chem. 249, 5889 (1974).</li><li>Lambert, J. P., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+proteins+and+DNA--proteomic+role+in+the+field+of+chromatin+research.">Of proteins and DNA--proteomic role in the field of chromatin research.</a> Mol. Biosyst. 6, 30 (2010).</li><li>Wu, C. Y., Jeng, K. S., Lai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SUMOylation+of+matrix+protein+M1+modulates+the+assembly+and+morphogenesis+of+influenza+A+virus.">The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus.</a> J. Virol. 85, 6618 (2011).</li><li>Liao, T. L., Wu, C. Y., Su, W. C., Jeng, K. S., Lai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination+of+NP+protein+regulates+influenza+A+virus+RNA+replication.">Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication.</a> EMBO J. 29, 3879 (2010).</li></ol>

No hay conflictos de interés declarado.

Aunque muchos estudios han identificado recientemente las proteínas individuales o de redes celulares implicados en la infección por el virus de la influenza 8, el significado funcional de la mayoría de estas interacciones aún no está claro. Dada la dependencia absoluta de la cromatina a base de funciones para la síntesis de ARN del virus de influenza y la compleja naturaleza biofísica y bioquímica del núcleo 9, nuevas técnicas se requieren para dilucidar estas funciones. El fraccionamien...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DMEM-alta glucosa </td><td> Gibco </td><td> 11965-092 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A9418 </td><td></td></tr><tr><td> Inhibidor de la proteasa mezcla G </td><td> Serva </td><td> 39101 </td><td></td></tr><tr><td> Benzonase nucleasa </td><td> Novagen </td><td> 71206 </td><td> 25 U / l </td></tr><tr><td> DNasa I, libre de RNasa </td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U / l </td></tr><tr><td> Homogeneizador Dounce </td><td> Wheaton </td><td> 432-1271 </td><td> Utilice el tipo &quot;B&quot; maja </td></tr><tr><td> Strep-Tactin Sepharose </td><td> IBA GmbH </td><td> 2-1201-025 </td><td> 50% formato de columna suspensión también se puede utilizar </td></tr><tr><td> Destiobiotina </td><td> IBAGmbH </td><td> 2-1000-002 </td><td></td></tr></tbody></table>

affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Como todos los virus negativo-hebra de ARN, el genoma de los virus de influenza está empaquetado en forma de complejos de ribonucleoproteína (vRNP virales), en los que se encapsidado el genoma de cadena simple por la nucleoproteína (NP), y asociado con el complejo de polimerasa trimérica consistente de la PA, PB1, PB2 y subunidades. Sin embargo, en contraste con la mayoría de los virus ARN, virus de la influenza realizar la síntesis de ARN viral en los núcleos de las células infectadas. Interesantemente, la síntesis de ARNm viral utiliza celulares pre-ARNm como cebadores, y se ha propuesto que este proceso se lleva a cabo en la cromatina 1. Las interacciones entre la polimerasa viral y el huésped ARN polimerasa II, así como entre NP y nucleosomas huésped también se han caracterizado 1,2. Recientemente, la generación de virus de la influenza recombinante que codifican un Uno-estrep-Tag genéticamente fusionado al extremo C-terminal de la subunidad PB2 de la polimerasa viral (RWSN-PB2-Strep 3) ha seres descrito. Estos virus recombinantes permite la purificación de PB2 que contienen complejos, incluyendo vRNPs, desde las células infectadas. Para obtener purificada vRNPs, cultivos de células infectadas, y vRNPs son purificados por afinidad a partir de lisados ​​derivados de estas células. Sin embargo, los procedimientos de lisis utilizado hasta la fecha se han basado en un paso de lisis detergente, que, a pesar de la presencia de una nucleasa general, a menudo extraer cromatina determinada único material ineficientemente. Nuestro trabajo preliminar sugiere que una gran parte de vRNPs nucleares no se extrajeron durante la lisis celular tradicional, y por lo tanto no podría ser purificado por afinidad. Para aumentar esta eficiencia de la extracción, y para separar la cromatina se dirigía de no cromatina determinada vRNPs nucleares, hemos adaptado un protocolo de paso a paso la extracción subcelular de la influenza células infectadas por virus. En pocas palabras, este procedimiento primero separa los núcleos de la célula y, a continuación extractos solubles proteínas nucleares (aquí denominado &quot;nucleoplasmic&quot; fracción).El restante material nuclear insoluble se digiere con Benzonase, un ADN inespecífica / ARN nucleasa, seguido de dos pasos de extracción de sal: en primer lugar utilizando 150 mM de NaCl (denominado &quot;CH150&quot;), a continuación, NaCl 500 mM (&quot;ch500&quot;) (fig. 1 ). Estos pasos de extracción de sal fueron elegidos en base a nuestra observación de que 500 mM de NaCl fue suficiente para solubilizar el 85% de vRNPs nucleares y aún así permitir la unión de vRNPs etiquetados a la matriz de afinidad. Después de fraccionamiento subcelular de las células infectadas, es posible purificar por afinidad vRNPs PB2-etiquetados de cada fracción individual y analizar su proteína y componentes de ARN utilizando Western Blot y extensión del cebador, respectivamente. Recientemente, hemos utilizado este método para descubrir que las exportaciones vRNP forma de complejos en los puntos finales después de la infección en la fracción de la cromatina se extrajo con 500 mM de NaCl (ch500) 3.

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Los virus de influenza replicar su genoma de ARN, en asociación con la cromatina de la célula huésped. A continuación, presentamos un método para purificar intactas ribonucleoprotein complejos virales de la cromatina de las células infectadas. Purificada complejos virales pueden ser analizados por tanto Western blot y extensión del cebador de contenido de proteínas y ARN, respectivamente.

Purificación por afinidad de los complejos de ribonucleoproteína Influenza Virus de la cromatina de las células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

affinity-purification-influenza-virus-ribonucleoprotein-complexes

Research

JoVE Journal

Immunology and Infection

13.0K vistas.  Universitätsklinikum Freiburg. Los virus de influenza replicar su genoma de ARN, en asociación con la cromatina de la célula huésped. A continuación, presentamos un método para purificar intactas ribonucleoprotein complejos virales de la cromatina de las células infectadas. Purificada complejos virales pueden ser analizados por tanto Western blot y extensión del cebador de contenido de proteínas y ARN, respectivamente.

Video: Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Generación de recombinantes del virus de influenza a partir del ADN plásmido

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Investigación

Inmunología e Infección

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

De alto rendimiento Método de detección de virus de la influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

La purificación del patógeno a partir de vacuolas<em&gt; Legionella</em&gt; Infectados por fagocitos

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

Expresión de funcionales recombinante hemaglutinina y neuraminidasa Proteínas del Virus Novel H7N9 Influenza usando el sistema de expresión de baculovirus

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagación del virus de la influenza en embrionados de pollo Huevos

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-inmunoprecipitación de la proteína de ratón Mx1 con la Influenza A nucleoproteína del virus

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Nasal Toallitas para Influenza A Detección de Virus y aislamiento de los cerdos

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

La purificación del ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; En los macrófagos

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

El análisis fenotípico y funcional de las células reguladoras T activadas Aislado de la coriomeningitis linfocítica crónica por el virus ratones infectados

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Determinación de Biofilm Iniciación, el virus de las células infectadas por bacterias y hongos

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Purificación por afinidad de los complejos de ribonucleoproteína Influenza Virus de la cromatina de las células infectadas

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