Gli autori desiderano ringraziare il Dr. James (Zhijian) Chen (UT Southwestern, Biologia Molecolare) per la fornitura di reagenti essenziali, tra cui i Mavs - / - topi, e il dottor Ren Sun (University of California-Los Angeles, Farmacologia e Medicina Molecolare ) per fornire il cromosoma artificiale batterico di γHV68 per questo studio.

1. Mouse con infezione γHV68 <ol><li> Topi cucciolata di sei a otto settimane di età, di genere abbinato (da 8 a 12 topi / gruppo) sono utilizzati per l&#39;infezione virale. Lasciare topi per ambientarsi nel corso di quattro giornate (96 ore) dopo la spedizione. </li><li> Passi del protocollo che utilizzano virus deve essere effettuata in un armadio di livello di biosicurezza 2 (BSL2) utilizzando le normali precauzioni BSL2. </li><li> Preparare sospensione virale (da 40 a 1 x 10 5 unità formanti placca [PFU]) di γHV68 in 30 ml di PBS sterile per topo poco prima dell&#39;esperimento. Tenere sospensione virale su ghiaccio. </li><li> Preparare la soluzione di ketamina / xilazina (1,5 ketamina mg e 0,15 mg xylazine/20 g di peso corporeo, 100 pl / mouse) per la sedazione del mouse. Assicurarsi che i topi sono stati sedati eseguendo un pizzico punta. </li><li> Topi sedate con 1,5 mg di ketamina e 0,15 mg xilazina per 20 mg di peso corporeo (100 pl / mouse) da intra-peritoneale di iniezione. </li><li> Deliver sospensione virale (30 microlitri / mouse) intranasale, in una goccia a gocciamoda, a una narice dei topi sedati. </li><li> Lay mouse su un lato per 5 - 10 minuti per facilitare la fornitura delle vie aeree del virus nei polmoni. </li><li> Luogo topi indietro nei topi in gabbia e il monitor fino a quando non sono completamente recuperati dalla sedazione. </li><li> A diversi giorni dopo l&#39;infezione, il sacrificio topi CO 2 asfissia e raccogliere i polmoni dopo aver assicurato la morte / perdita di coscienza profonda. Polmoni Luogo in sterili da 1,5 ml con tappo a vite tubi contenenti 500 ml di 1,0 millimetri Zirconia / silice perline. Tenere i tubi in ghiaccio. Conservare i campioni a -80 ° C se non di procedere alla fase successiva nello stesso giorno. La milza o tessuto epatico potrebbero essere raccolti in questo momento per l&#39;analisi della latenza virale seconda temporale dell&#39;esperimento. </li><li> Aggiungere 1 ml di freddo DMEM privo di siero nella provetta e omogeneizzare i polmoni dal tallone-pestaggio 30 secondi. Raffreddare provette in ghiaccio per almeno 1 min. Ripetere questa operazione una sola volta. </li><li> Polmone lisati centrifuga a 16.000 rcf a 4 ° C per 1 min e utilizzare supernatant per determinare il titolo virale da un saggio di placca con un NIH3T3 o BHK21 monostrato (si veda la Sezione 5 per i dettagli). </li></ol> 2. Determinare γHV68 multi-step cinetica di crescita in fibroblasti embrionali di topo <ol><li> Crescere tipo selvatico fibroblasti embrionali di topo (MEF) e quelli carenti in un gene host sub-confluenti (circa 80%) densità prima placcatura cellule. </li><li> Suddiviso in MEFs 24 pozzetti a 10.000 cellule / pozzetto per una bassa multipilicity-di-infezione (MOI) il giorno prima dell&#39;infezione. Gli esperimenti sono normalmente effettuate in triplicato e ad una MOI tra 0,001-0,05 viene usato solitamente. </li><li> Preparare γHV68 sospensione contenente quantità desiderata di virus (0,5 ml per pozzetto). </li><li> Estrarre il supporto di MEF e coprire con 0,5 ml di sospensione γHV68 per pozzetto. </li><li> Incubare la piastra in coltura tissutale incubatore, roccia ogni 30 min, e consentono di procedere incubazione per 2 ore. </li><li> Rimuovere sospensione virale, e coprire le cellule con 0,5 ml di fresca complete DMEM terreno contenente 8% di siero fetale bovino. </li><li> A diversi giorni dopo l&#39;infezione, raccogliere il medio e cellule in provette sterili 1,5 ml per centrifuga. Subito congelare provette a -80 ° C. </li><li> Rilasciare γHV68 da MEF mediante congelamento a -80 ° C, lo scongelamento a 37 ° C bagno di acqua e vortex. Tre cicli di congelamento e scongelamento viene solitamente applicata ai campioni. </li><li> Determinare titolo virale da un saggio di placca con un NIH3T3 o BHK21 monostrato (si veda la Sezione 5 per i dettagli). </li><li> Leggi il titolo virale e la trama γHV68 multi-step curva di crescita sul MEF. </li></ol> 3. Dissezione molecolare dei γHV68 replicazione litica in fibroblasti embrionali di topo <ol><li> Eseguire infezione virale, come descritto nei punti da 2.1 a 2.6 della sezione 2. </li><li> A diversi giorni dopo l&#39;infezione, scartare il surnatante. Lavare le cellule con PBS freddo e le cellule Tripsinizzare. Cellule pellet per centrifugazione a 1.000 rcf a temperatura ambiente per 1 min. Eliminare il supernatante ecellule conservare a -80 ° C. </li><li> Estrarre DNA totale (host e del genoma virale) e l&#39;RNA totale secondo i metodi precedentemente pubblicati 5,6. </li><li> Eseguire in tempo reale PCR utilizzando DNA totale e primer specifici per trascritti virali litici come RTA (ORF50), ORF57 e ORF60. Determinare la quantità relativa di intracellulare γHV68 genoma in riferimento ad un gene housekeeping host (ad esempio, β-actina). </li><li> Per rimuovere la contaminazione di DNA genomico, il trattamento con RNasi-free DNasi è fondamentale per la preparazione di cDNA con RNA totale e oligo (dT) primer 11-19. Fare riferimento al riferimento 5 per maggiori dettagli in materia di estrazione di RNA tra cui il trattamento con RNasi-free DNasi e RT-PCR. Eseguire in tempo reale PCR utilizzando primer cDNA e come sopra per determinare la quantità relativa di trascritti virali in riferimento a quella del gene housekeeping ospitante. </li></ol> 4. Generazione ricombinante γHV68 con cromosoma batterico artificiale La metodologiad qui descritto viene utilizzato per introdurre mutazioni in un gene γHV68 che è coinvolto nella interazione ospite-virus. <ol><li> Preparare un frammento di DNA (circa 1,5 kb) di sequenza wild-type o sequenza portando mutazioni desiderate nella regione centrale mediante PCR. </li><li> Preparare cromosoma artificiale batterico (BAC) che contiene un inserimento trasposone 7 attorno al sito di mutazione, che inattiva il gene specifico gene di interesse, da midi scala purificazione (Origene). Conservare BAC DNA a 4 ° C (evitare di gelo / disgelo del BAC DNA). </li><li> Transfettare BAC DNA e prodotto di PCR contenente mutazioni desiderate del gene di interesse in cellule (ad esempio, o NIH3T3 BHK21), che sono altamente permissive per la replicazione γHV68 litico, con Lipofectamine 2000 (Invitrogen). </li><li> Mantenere la divisione delle celle fino effetto citopatico (CPE) presenta in monostrato. Raccogliere contenenti virus surnatante e, se necessario, amplificare virus in cellule NIH3T3 o BHK21. </li><li> Infettare cellule NIH3T3 convirus ricombinante mediante centrifugazione a 1800 rpm, 30 ° C per 30 min. </li><li> Harvest γHV68 infettate cellule NIH3T3 e preparare genoma virale circolarizzato Hirt utilizzando il protocollo di 8,9. </li><li> Electro-MAX trasformare cellule competenti DH10B (Invitrogen) per elettroporazione e schermo per colonie che sono la resistenza al cloramfenicolo (Cm), ma sensibile alla kanamicina (kan) (Cm gene resistente è in backbone BAC, mentre Kan gene resistente è in trasposone inserimento). </li><li> Grow Cm R S Kan colonie in terreno contenente chlorophenicol e preparare BAC DNA con la purificazione o mini-midi-scala (Origene). </li><li> Verificare la mutazione desiderata nel gene bersaglio mediante PCR, utilizzando primer specifici per le regioni fiancheggianti del sito di mutazione, e sequenziamento. </li><li> Confermare non riarrangiamento cromosomico in BAC mediante digestione con enzimi di restrizione selezionati e impulsi campo elettroforesi su gel. </li><li> Transfettare BAC ricombinante in cellule NIH 3T3 o BHK21 con Lipofectamine2000 (Invitrogen) per preparare γHV68 ricombinante. </li><li> Tenere passaging cellule fino a CPE si presenta in monostrato. Raccogliere virus contenente surnatante e amplificare ricombinante γHV68 in NIH3T3 o BHK21 cellule. </li><li> Determinare il titolo del virus ricombinante e caratterizzare la crescita virale proprietà ex vivo e in vivo, come descritto nelle sezioni 1 e 2. </li></ol> 5. Determinare il titolo virale da un saggio di placca <ol><li> Crescere cellule NIH3T3 di sub-confluenti (circa 80%) densità prima placcatura cellule. </li><li> Diviso NIH3T3 in 24 pozzetti a 20.000 cellule / pozzetto il giorno prima dell&#39;infezione. </li><li> Preparare 10 volte supernatanti virus in serie diluiti con DMEM terreno contenente l&#39;8% di siero di vitello neonato (NCS). </li><li> Rimuovere medie e coprire cellule NIH3T3 con 0,5 ml di sospensione γHV68. </li><li> Incubare la piastra in incubatore tessuto, oscillare ogni 30 min, e consentono di procedere incubazioneper 2 h. </li><li> Rimuovere sospensione virale e cellule copertura con 0,5 ml di terreno DMEM contenente il 2% e 0,75% NCS metilcellulosa (Sigma). </li><li> Incubare la piastra in incubatore per colture dei tessuti. Contare placche al giorno 6 post-infezione. Colorazione del monostrato, ad esempio con violetto cristallo, può facilitare conteggio placca. </li><li> Calcolare il titolo virale nei lisati di tessuto non diluiti o lisati cellulari usando la seguente formula: Titer (PFU / ml) = D x N x 1000 microlitri / ml V ÷ microlitri. N, il numero medio di placca ad una diluizione appropriata, D, diluizione volte (ad esempio 5, 10, 100 .....) V (pl), volume di supernatante-diluito serialmente aggiunti per pozzetto. </li></ol> 6. Rappresentante dei risultati: Tre figure rappresentative sono mostrati qui, tra cui la replica γHV68 litico nel polmone di wild-type e Mavs - / - topo 10, γHV68 fenotipi replica litici in fibroblasti embrionali di topo (MEF), e recombinformica γHV68 portatori di mutazioni nei siti di fosforilazione che sono modulati dal MAV-dipendente IKKβ. Questi tre esperimenti concordanti costituiscono un sistema per definire i ruoli dei componenti del sistema immunitario innato in γHV68 replicazione litica in vivo ed ex vivo. <img alt="Figura 1" src="/files/ftp_upload/3140/3140fig1.jpg" /> Figura 1 γHV68 carichi nei polmoni dei Mavs + / + e Mavs -. / - Topi. A) a due vie di segnalazione principale innata a valle del MAV. I Mavs adattatore relè molecola di segnalazione da citosolici RIG-I-simili recettori per attivare NFκB e fattori interferone regolamentazione (IRF) che, a sua volta, up-regolare l&#39;espressione genica di citochine proinfiammatorie e gli interferoni. B) Mavs + / + e Mavs - / - topi sono stati infettati con il 40 PFU γHV68 intranasale e carica virale nel polmone a tempi indicati, sono stati deter<html> estratto da un saggio di placca usando NIH3T3 monostrato. Ogni simbolo rappresenta un mouse. <img alt="Figura 2" src="/files/ftp_upload/3140/3140fig2.jpg" /> Figura 2. ΓHV68 cinetica replica litici in topo fibroblasti embrionali (MEF). La replicazione litica di γHV68 su Mavs + / + e Mavs - / - MEF sono state esaminate dalla multi-step curve di crescita (A) e quantitativa real-time PCR (B). Per entrambi gli esperimenti, pari numero di MEFs e quantità di γHV68 stati usati per infezione virale ad una molteplicità d&#39;infezione (MOI) di 0,01. (A) cellule e dei surnatanti sono stati raccolti a tempi indicati e soggetti ad un saggio di placca per determinare i titoli virali. (B) L&#39;RNA totale è stato estratto da γHV68 infetto MEFs e analizzati mediante real time PCR con primer specifici per determinati trascritti litico (RTA, ORF57, ORF60 e). ig3.jpg &quot;/&gt; Figura 3. La cinetica di replica litici di ricombinanti γHV68 mutazioni che trasportano all&#39;interno del dominio di transattivazione RTA che aboliscono fosforilazione da IKKγ. (A) Multi-step curve di crescita ricombinante di virus wild-type (γHV68.wt) e virus mutante (γHV68.mut) in Mavs + / + e Mavs - / - cellule MEF (MOI = 0,01). MEFs stati infettati con γHV68 ad una MOI di 0,01. Cellule e dei surnatanti sono stati raccolti a tempi indicati e titoli virali sono state determinate mediante un saggio di placca usando monostrato NIH 3T3. (B) γHV68 RTA livello di mRNA in γHV68 infettate cellule NIH3T3 (MOI = 0,01). A 30 h post-infezione, l&#39;RNA totale è stato estratto da cellule infettate γHV68 NIH 3T3 e analizzati mediante quantitativa PCR in tempo reale.

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+microorganisms+and+activation+of+the+immune+response.">Recognition of microorganisms and activation of the immune response.</a> Nature. 449, 819-826 (2007).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Speck, S. H., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+latency+and+its+regulation:+lessons+from+the+gamma-herpesviruses.">Viral latency and its regulation: lessons from the gamma-herpesviruses.</a> Cell Host Microbe. 8, 100-115 (2010).</li><li>Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. 6, e1001001-e1001001 (2010).</li><li>Strauss, W. M., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+genomic+DNA+from+mammalian+tissues.">Preparation of genomic DNA from mammalian tissues.</a> Current Protocols in Molecular Biology. , 2-2 (1998).</li><li>Song, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+viral+genes+essential+for+replication+of+murine+gamma-herpesvirus+68+using+signature-tagged+mutagenesis.">Identification of viral genes essential for replication of murine gamma-herpesvirus 68 using signature-tagged mutagenesis.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 3805-3810 (2005).</li><li>Hirt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures.">Selective extraction of polyoma DNA from infected mouse cell cultures.</a> J. Mol. Biol. 26, 365-369 (1967).</li><li>Eva-Maria Borst, E., Crnkovic-Mertens, I., Messerle, M., Zhao, S., Stodolsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+&#946;-herpesvirus+genomes+as+bacterial+artificial+chromosomes.">Cloning of &#946;-herpesvirus genomes as bacterial artificial chromosomes.</a> Methods in Molecular Biology. , 256-256 (2004).</li><li>Sun, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Meylan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Xu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

In risposta ad un&#39;infezione virale, i Mavs-dipendenti immunitarie innate vie di segnalazione sono attivati ​​per promuovere la produzione di citochine infiammatorie antivirali 10-14. Utilizzando murino γHV68 come un virus modello per umano oncogenico sarcoma di Kaposi associato herpesvirus e virus di Epstein-Barr 3,4, abbiamo scoperto che γHV68 usurpa i Mavs-IKKβ della via per promuovere la replicazione virale litico attraverso l&#39;attivazione trascrizionale 5. Impiegando ge...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-019 </td></tr><tr><td> Electro-MAX cellule DH10B competenti </td><td> Invitrogen </td><td> 18290-015 </td></tr><tr><td> Metilcellulosa </td><td> Sigma </td><td> M0512 </td></tr><tr><td> POWERPREP HP plasmide sistema Miniprep </td><td> Origene </td><td> NP100004 </td></tr><tr><td> POWERPREP HP plasmide sistema Midiprep </td><td> Origene </td><td> NP100006 </td></tr></tbody></table>

dissecting host-virus interaction in lytic replication of a model herpesvirus

In risposta ad un&#39;infezione virale, un ospite sviluppa varie risposte difensive, come l&#39;attivazione immunitaria innata vie di segnalazione che portano alla produzione di citochine 1,2 antivirale. Al fine di colonizzare l&#39;ospite, i virus sono obbligati a sottrarsi risposte dell&#39;ospite antivirali e manipolare vie di segnalazione. Svelare l&#39;ospite-virus interazione farà luce sullo sviluppo di nuove strategie terapeutiche contro le infezioni virali. Murine γHV68 è strettamente correlata alla umana oncogeno sarcoma di Kaposi associato herpesvirus e Epsten-Barr 3,4. γHV68 infezione nei topi di laboratorio fornisce un modello trattabile piccolo animale per esaminare l&#39;intero corso di risposte dell&#39;ospite e infezione virale in vivo, che non sono disponibili per herpesvirus umani. In questo protocollo, vi presentiamo un gruppo di metodi per la caratterizzazione fenotipica e la dissezione molecolare dei componenti host di segnalazione in γHV68 Replica liticozione sia in vivo ed ex vivo. La disponibilità di ceppi di topi geneticamente modificati permette l&#39;interrogazione dei ruoli di vie di segnalazione di accoglienza durante γHV68 infezione acuta in vivo. Inoltre, fibroblasti embrionali di topo (MEF) isolati da questi ceppi di topi deficienti può essere utilizzato per analizzare ulteriormente ruoli di queste molecole durante γHV68 litico ex vivo replica. Utilizzo di test virologici e biologia molecolare, siamo in grado di individuare il meccanismo molecolare di ospite-virus e identificare le interazioni ospite e geni virali essenziali per la replicazione virale litico. Infine, un cromosoma artificiale batterico (BAC) sistema facilita l&#39;introduzione di mutazioni nel fattore virale (s) che specificamente interrompere l&#39;interazione virus-ospite. Ricombinante γHV68 portare queste mutazioni possono essere utilizzati per ricapitolare i fenotipi di γHV68 replicazione litica in MEF deficienti di accoglienza chiave componenti di segnalamento.Questo protocollo offre un&#39;ottima strategia per interrogare interazione ospite-patogeno a più livelli di intervento in vivo e ex vivo. Di recente, abbiamo scoperto che γHV68 usurpa un pathway di segnalazione immunitaria innata per promuovere replicazione virale litico 5. In particolare, γHV68 de novo infezione attiva il sistema immunitario IKKβ chinasi e IKKβ attivato fosforila il fattore di trascrizione virale maestro, replica e transattivatore (RTA), per la promozione virale attivazione trascrizionale. In tal modo, γHV68 efficiente coppie sua attivazione trascrizionale di ospitare attivazione immunitaria innata, facilitando così la trascrizione e la replicazione virale litico. Questo studio fornisce un esempio eccellente che può essere applicato ad altri virus per interrogare host-virus interazione.

Watch this Scientific Journal Video about Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus at JoVE.com

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Descriviamo un protocollo per identificare i ruoli chiave di molecole di segnalazione di accoglienza nella replicazione litica di un herpesvirus modello, gamma herpesvirus 68 (γHV68). Utilizzando ceppi di topi geneticamente modificati e fibroblasti embrionali per γHV68 replica litico, il protocollo consente sia la caratterizzazione fenotipica e molecolare del virus interrogatorio interazioni ospite-in replicazione virale litico.

Dissezione Host-virus Interazione in replica litico di un Herpesvirus modello

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

dissecting-host-virus-interaction-lytic-replication-model

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JoVE Journal

Immunology and Infection

10.8K Views.  UT Southwestern Medical Center. Descriviamo un protocollo per identificare i ruoli chiave di molecole di segnalazione di accoglienza nella replicazione litica di un herpesvirus modello, gamma herpesvirus 68 (γHV68). Utilizzando ceppi di topi geneticamente modificati e fibroblasti embrionali per γHV68 replica litico, il protocollo consente sia la caratterizzazione fenotipica e molecolare del virus interrogatorio interazioni ospite-in replicazione virale litico.

Video: Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificare i geni disregolazione indotta da sarcoma di Kaposi associato Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

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Immunologia e infezioni

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissezione Innate Immune Signaling in Evasion virale di produzione di citochine

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

High-throughput Assay per Fenotipo<em&gt; Salmonella enterica</em&gt; Typhimurium Associazione, Invasion, e replica nei macrofagi

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studiare comunità microbiche<em&gt; In Vivo</em&gt;: Un modello di interazione ospite-mediata tra<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nei Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis come organismo modello per la Valutazione Interazione di batteri anaerobici con cellule ospiti

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

Sviluppo e validazione di un metodo quantitativo per PCR Equid herpesvirus-2 Diagnostica in respiratorie Fluidi

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In Vitro</em&gt; Modello della barriera emato-encefalica Utilizzando spettroscopia di impedenza: un focus sui T Cell-endoteliale cellulare Interazione

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissezione multiproteici segnalazione di purificazione di affinità di complementazione bimolecolare (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.