Gli autori desiderano ringraziare Nada Naffakh e Marie-Anne Rameix-Welti (Institut Pasteur) per la rWSN-PB2-Strep virus.

<html> Un diagramma di flusso schematico del protocollo è mostrato in fig. 1 e una tabella di reagenti è presentato di seguito. 1. Infezione (16 - 24 h) <ol><li> Seed le cellule HeLa in 5 mm 150 piatti di plastica di coltura di cellule in terreno Dulbecco modificato Eagle (DMEM)-medio-alta di glucosio, in modo tale che arriveranno circa l&#39;80% di confluenza il giorno seguente (circa 5 x 10 8 cellule). È importante che le cellule non raggiunge il 100% di confluenza. </li><li> Il giorno successivo, diluire il Strep-tag stock di virus influenzale in tampone fosfato isotonico (PBS) contenente 0,3% di sieroalbumina bovina (BSA) ad una molteplicità di infezione di 3. </li><li> Rimuovere il mezzo di crescita delle cellule, lavare una volta con PBS, e aggiungere 10 ml di PBS contenente il virus alle cellule. Incubare per 1 ora a temperatura ambiente. </li><li> Sostituire il mezzo infezione con DMEM-alto glucosio, e continuare incubazione a 37 ° C. </li></ol>&quot;&gt; La durata dell&#39;incubazione dipende dalla fase dell&#39;infezione da studiare (vedi Discussione). 2. Frazionamento subcellulare (3 h) <ol><li> Lavare le cellule una volta con PBS freddo e poi sospendere le cellule in PBS freddo con un raschietto in gomma e il trasferimento ad un tubo da 50 ml. </li><li> Cellule pellet in una centrifuga da tavolo per 5 minuti a 1.000 rpm e quindi aspirare PBS. </li><li> Risospendere il pellet cellulare in 10 ml di tampone saccarosio freddo (10 mM HEPES pH 7,9, KCl 10 mM, 2 mM acetato di Mg (MgOAc), 3 mM CaCl 2, saccarosio 340 mM, 1 mM ditiotreitolo (DTT), 1% mix inibitore delle proteasi) . Dopo la sospensione, le cellule passano attraverso una punta di pipetta 200 microlitri attaccato ad una pipetta 10 ml di siero di interrompere grumi di cellule. </li><li> Incubare 10 minuti sul ghiaccio. Capovolgere periodicamente per risospendere le cellule. </li><li> Aggiungere Nonidet P-40 (NP-40) ad una concentrazione finale di 0,5% e vortice ad alta velocità per 15 sec. Immediatamente centrifugare per 10 min a 3500 xga 4 ° C in una centrifuga da tavologe. </li><li> Rimuovere il surnatante e conservare a 4 ° C. Questa è la frazione citoplasmatica. Il pellet deve essere più piccolo e più bianco il pellet di cellule originale. Tutte le frazioni possono essere conservate per almeno 4 ore a 4 ° C. </li><li> Lavare il pellet in 5 ml freddo tampone saccarosio, re-centrifuga come nel punto 2.5 e scartare il surnatante. </li><li> Risospendere il pellet (contenente nuclei) in 1,5 ml di tampone di estrazione a freddo nucleoplasmic (50 mM HEPES pH 7,9, 150 mM KOAc, 1,5 mM MgCl 2, 0,1% NP-40, 1 mM DTT, 1% mix inibitori delle proteasi). </li><li> Trasferire in una fredda, completamente in vetro, 4 ml Dounce omogeneizzatore con un aderente pestello e omogeneizzare i nuclei con 20 colpi. Evitare la formazione di schiuma durante omogeneizzazione. La resistenza dovrebbe aumentare dopo circa 10 colpi. Omogeneizzazione efficiente può essere confermata con un microscopio a contrasto di fase. </li><li> Trasferire la nuclei omogeneizzato in una provetta 1,5 ml microcentrifuga e incubare 20 min su una ruota girevole a 4 °C. </li><li> Centrifugare a 16.000 x g in una microcentrifuga a 4 ° C per 10 min. </li><li> Rimuovere il surnatante e conservare a 4 ° C. Questa è la frazione nucleoplasmic. </li><li> Risospendere il pellet in 1,5 ml di tampone di digestione (50 mM HEPES pH 7,9, 10 mM NaCl, 1,5 mM MgCl 2, 1 mM DTT, 1% mix inibitore della proteasi, 100 U / ml Benzonase (per analisi RNA, sostituirlo con 20 U / ml RNasi senza DNasi I)) pre-riscaldato a 37 ° C, e incubare 10 min a 37 ° C. </li><li> Aggiungere 42 microlitri di NaCl 5 M (per concentrazione finale di 150 mM NaCl) e incubare ulteriori 20 min a 4 ° C. </li><li> Centrifugare a 16.000 x g in una microcentrifuga a 4 ° C per 10 min. </li><li> Rimuovere il surnatante e conservare a 4 ° C. Questa è la frazione CH150. </li><li> Risospendere il pellet in 1,5 ml freddo alto sale tampone (50 mM HEPES pH 7,9, 500 mM NaCl, 1,5 mM MgCl 2, 0,1% NP-40, 1 mM DTT, 1% mix inibitore di proteasi) e incubare 20 min in ghiaccio. </li><li> Centrifugare a 16000 x g &lt;/ Em&gt; in una microcentrifuga a 4 ° C per 10 min. </li><li> Rimuovere il surnatante e conservare a 4 ° C. Questa è la frazione ch500. </li></ol> 3. Strep-tag Purificazione (2 h) <ol><li> Aggiungere 300 pl del 5 M NaCl alla frazione citoplasmatica (per concentrazione finale di 150 mM NaCl). </li><li> Aliquotare 100 pl di Strep-confezionati Tactin perle Sepharose per frazione in un tubo da 15 ml. </li><li> Aggiungere 10 volumi di tampone di lavaggio Strep (20 mM HEPES pH 7,9, 150 mM NaCl (500 mM NaCl per ch500 campioni), 1 mM EDTA) a perline, invertire la provetta per miscelare, e centrifugare 5 min a 1.000 x g in un tavolo centrifugare. </li><li> Eliminare il surnatante, ripetere il punto 3,3 due volte e poi risospendere il pellet tallone in un volume uguale di tampone di lavaggio Strep. </li><li> Aggiungere 200 microlitri di lavato Strep-Tactin impasto tallone per ogni frazione (in provette per microcentrifuga da 1,5 ml, i nuceloplasmic CH150 e ch500 frazioni e un tubo 15 ml conica per la frazione citoplasmatica). </li><li> Ruotareprovette 1h a 4 ° C su una ruota girevole. </li><li> Centrifugare provette 1 min a 1.000 x ga 4 ° C. Gettare il surnatante. </li><li> Aggiungere 1 ml di tampone di lavaggio a freddo Strep in ciascun tubo. Trasferire perline provetta da 15 ml (frazione citoplasmatica) a 1,5 ml provetta. Lavare tutti 1 min tubi a 4 ° C, dopo centrifugazione come nel passaggio 3,7. Ripetere questa fase di lavaggio due volte. </li><li> Dopo l&#39;ultimo lavaggio, rimuovere tutte le tracce di tampone di lavaggio con una punta piatta pipetta. Aggiungere 100 ul tampone di eluizione (tampone di lavaggio Strep contenente 2,5 mM desthiobiotin) e mescolare delicatamente. </li><li> Incubare 15 min in ghiaccio. Mescolare delicatamente di tanto in tanto perle toccando il fondo delle provette. </li><li> Provette per centrifuga 1 min a 1000 x ga 4 ° C in microcentrifuga. Raccogliere il supernatante contenente eluite Strep-tag vRNPs. </li></ol> 4. Risultati rappresentativi L&#39;efficienza del frazionamento è meglio giudicato mediante analisi Western blot di lisati frazionati utilizzando antibodies specifici per i marcatori subcellulari (Fig. 2). In particolare, successo frazionamento subnucleare dovrebbe mostrare poca o nessuna cellulare RNA polimerasi II (Pol II) in nucleoplasmic, o frazione solubile nucleare 4, e più devono essere estratti con 150 mM NaCl 5. Un degrado di Pol II è riproducibile osservata in influenzali cellule infettate da virus rispetto alle cellule non infette, in linea con la letteratura 6. La purificazione di vRNPs è meglio giudicato da argento o colorazione con Coomassie, come mostrato per un eluato citoplasmatica in Fig. 3. Nella nostra esperienza, la maggior parte Strep-PB2 viene purificato come parte di un mercato completamente formata vRNP, vale a dire polimerasi poco solubile viene catturato. Ciò si riflette in alto NP: PA/PB1/PB2 rapporto osservato argento o colorazione Coomassie. Un rapporto inferiore suggerisce la contaminazione tampone con RNasi, come diverse RNasi onnipresenti (come RNasi A) sono noti a RNA virale clivare in modo tale da dissocimangiato la polimerasi dalla NP multimeri 7. Interessante, il trattamento con BenzonaseTM sola, mentre sufficiente a digerire RNA virale, sembra non avere alcun effetto sui rapporti stechiometrici di proteine ​​vRNP. Anche se non possiamo spiegare questo fenomeno, abbiamo osservato interruzione delle vRNPs dopo digestione con RNasi altri dati non mostrati), suggerendo che Benzonase può lasciare alcune regioni strutturalmente importanti RNA intatto. Dopo purificazione del vRNPs dal citoplasma e nucleoplasma (eseguita senza digestione nucleasi), 8 deboli bande discrete ma ad alti pesi molecolari può essere osservato da colorazione con argento, che corrispondono ai pesi molecolari previsti dei segmenti genomici influenza 8 (vedi rif. 3 ). Le quantità relative di vRNPs purificati da ogni frazione a 9 h dopo l&#39;infezione sono mostrati nella fig. 4. La distribuzione di vRNPs varia nel corso dell&#39;infezione, con forte accumulo nella frazione CH150 in punti temporali iniziali, eaumentato accumulo nel nucleoplasma e citoplasma in punti temporali tardivi. <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso del frazionamento subcellulare. Adattato da ref. 3. <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="Figura 2"/> Figura 2. Analisi Western blot delle frazioni subcellulari da influenza cellule infettate da virus. 10 9 cellule HeLa sono state infettate con WSN ceppo del virus influenzale per 9 ore prima di frazionamento subcellulare. Uguali quantità di proteine ​​totali sono state caricate in ciascuna corsia e analizzati utilizzando gli anticorpi mostrate. RNA polimerasi II (Pol II) è stato rilevato usando clone 8WG16, che riconosce tutte le forme del dominio C-terminale (la banda ipofosforilata è più prominente). Adattato da ref. 3. <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="Figura 3"/> Figura 3.Argento analisi macchia di vRNPs purificata. Cellule HeLa sono state infettate con rWSN (come controllo negativo) o rWSN-PB2-Strep per 9 h, seguito da purificazione Strep dalla frazione citoplasmatica. Asterisco indica le bande che non sono visibili dopo la digestione RNasi. Adattato da ref. 3. <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="Figura 4"/> Figura 4. Argento analisi macchia di vRNPs purificati da diverse frazioni cellulari. 10 9 cellule HeLa sono state infettate con rWSN-PB2-Strep o rWSN per 9 h, seguito da frazionamento subcellulare e purificazione Strep. Uguali quantità di eluato da ogni frazione sono stati caricati. Adattato da ref. 3.

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	<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. 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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. 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Mentre molti studi hanno recentemente identificato singole proteine ​​o reti cellulari coinvolti nella infezione da virus dell&#39;influenza 8, il significato funzionale della maggior parte di queste interazioni è ancora chiaro. Data la dipendenza assoluta di cromatina basati funzioni per la sintesi di RNA del virus influenzale e la natura complessa biofisica e biochimica del nucleo 9, nuove tecniche saranno necessari per chiarire tali funzioni. Il frazionamento subnucleare che presentiamo qui...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> DMEM-alto glucosio </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> Inibitore della proteasi Mix G </td><td> Serva </td><td> 39101 </td><td></td></tr><tr><td> Benzonase nucleasi </td><td> Novagen </td><td> 71206 </td><td> 25 U / pl </td></tr><tr><td> I DNasi, RNasi-free </td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U / pl </td></tr><tr><td> Dounce omogeneizzatore </td><td> Wheaton </td><td> 432-1271 </td><td> Utilizzare digitare pestello &quot;B&quot; </td></tr><tr><td> Strep-Tactin Sepharose </td><td> IBA GmbH </td><td> 2-1201-025 </td><td> 50% formato colonna sospensione può anche essere usato </td></tr><tr><td> Desthiobiotin </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

Come tutti filamento negativo-virus ad RNA, il genoma di virus influenzali viene confezionato in forma di complessi ribonucleoproteina virali (vRNP), in cui è incapsidati singolo filamento genoma della nucleoproteina (NP), e associate con il complesso polimerasi composto trimerico della PA, PB1, e PB2 subunità. Tuttavia, contrariamente alla maggior parte dei virus a RNA, virus dell&#39;influenza eseguire sintesi di RNA virale nei nuclei delle cellule infette. È interessante notare, sintesi di mRNA virale utilizza cellulari pre-mRNA come primer, ed è stato proposto che questo processo ha luogo sulla cromatina 1. Le interazioni tra la polimerasi virale e l&#39;host RNA polimerasi II, nonché tra NP e nucleosomi ospitanti sono stati caratterizzati 1,2. Recentemente, la generazione di virus influenzali ricombinanti codificanti una One-Strep-Tag geneticamente fuso al C-terminale della subunità PB2 della polimerasi virale (rWSN-PB2-Strep 3) ha essereen descritto. Questi virus ricombinanti permettono la purificazione di PB2 contenenti complessi, compresi vRNPs, da cellule infette. Per ottenere purificato vRNPs, colture cellulari sono infetti, e vRNPs sono purificati per affinità da lisati derivati ​​da queste cellule. Tuttavia, le procedure di lisi utilizzati fino ad oggi sono stati basati su una fase di lisi detersivo, che, nonostante la presenza di una nucleasi generale, spesso estrarre cromatina materiale legato solo inefficiente. Il nostro lavoro preliminare suggerito che una grande porzione di vRNPs nucleari non sono stati estratti durante la lisi cellulare tradizionale, e quindi non poteva essere purificato per affinità. Per aumentare questa efficienza di estrazione, e per separare cromatina-legato da vRNPs nucleari non-cromatina-bound, abbiamo adattato una graduale protocollo subcellulare di estrazione influenza cellule infettate da virus. In breve, questa procedura prima separa i nuclei dalla cella e poi estratti solubili proteine ​​nucleari (qui chiamato frazione &quot;nucleoplasmic&quot;).Il rimanente materiale insolubile nucleare viene poi digerito con BenzonaseTM, un DNA aspecifica / RNA nucleasi, seguita da due passi sale di estrazione: prima utilizzando 150 mM NaCl (denominato &quot;CH150&quot;), allora 500 mM NaCl (&quot;ch500&quot;) (Fig. 1 ). Queste fasi di estrazione del sale sono stati scelti sulla base della nostra osservazione che 500 mM NaCl è stato sufficiente a solubilizzare oltre l&#39;85% del vRNPs nucleari ma ancora consentire il legame degli vRNPs tag alla matrice di affinità. Dopo frazionamento subcellulare di cellule infette, è possibile purificare affinità vRNPs PB2-etichetta da ogni frazione individuo e analizzare le proteine ​​e loro componenti RNA utilizzando Western Blot e la estensione di primer, rispettivamente. Recentemente, abbiamo utilizzato questo metodo per scoprire che vRNP forma export complessi durante i punti in ritardo dopo l&#39;infezione sulla frazione cromatina estratta con 500 mM 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

I virus influenzali replicare il loro genoma RNA in associazione con la cellula ospite cromatina. Qui, vi presentiamo un metodo per purificare intatti complessi ribonucleoproteina virali dalla cromatina delle cellule infette. Purificato complessi virali possono essere analizzati mediante Western blot e sia di estensione del primer di proteine ​​e RNA, rispettivamente.

Purificazione per affinità di Complessi virus influenzali ribonucleoproteina dalla cromatina delle cellule infettate

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

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Immunology and Infection

13.0K Views.  Universitätsklinikum Freiburg. I virus influenzali replicare il loro genoma RNA in associazione con la cellula ospite cromatina. Qui, vi presentiamo un metodo per purificare intatti complessi ribonucleoproteina virali dalla cromatina delle cellule infette. Purificato complessi virali possono essere analizzati mediante Western blot e sia di estensione del primer di proteine ​​e RNA, rispettivamente.

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.

Generazione di influenza da virus ricombinante del DNA plasmidi

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

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Immunologia e infezioni

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.

High-throughput Metodo di rilevazione per il virus dell&#39;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.

Purificazione di Vacuoli patogeno da<em&gt; Legionella</em&gt;-Infetti fagociti

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.

Espressione dei funzionali ricombinante emoagglutinina e neuraminidasi proteine ​​del virus Novel H7N9 Influenza utilizzando l&#39;espressione sistema 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.

Influenza Virus Propagazione in embrionate di pollo Uova

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-immunoprecipitazione della proteina mouse Mx1 con il virus nucleoproteina Influenza A

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.

Panni nasale per l&#39;influenza A rilevazione antivirus e isolamento da Suina

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

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.

Fenotipica e analisi funzionale di cellule T regolatorie attivati ​​Isolato dal linfocitica cronica coriomeningite topi infettati da virus

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.

Determinazione del biofilm iniziazione su cellule infettate da virus da batteri e funghi

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

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generazione di varianti di fuga di neutralizzare l'Influenza Virus anticorpi monoclonali

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...